KR20240038723A - CAR-expressing immune cells specifically targeting mesothelin and uses thereof - Google Patents

Abstract

Disclosed herein are engineered immune cells that specifically recognize mesothelin and express IL-15 and optionally CCL19. Additionally, an antibody that specifically recognizes mesothelin, and a polynucleotide encoding a chimeric antigen receptor (CAR) comprising the 4-1BB intracellular region; and a polynucleotide encoding IL-15; and, optionally, an isolated nucleic acid molecule comprising a polynucleotide encoding CCL19, vectors, pharmaceutical compositions comprising the nucleic acid molecule, and methods of using engineered immune cells.

Description

CAR-expressing immune cells specifically targeting mesothelin and uses thereof

Cross-reference to related applications

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63/306,862, filed February 4, 2022, and U.S. Provisional Application No. 63/227,115, filed July 29, 2021, which It is incorporated herein by reference in its entirety.

technology field

The present invention relates to a cell surface molecule specifically recognizing human mesothelin, immune cells expressing interleukin 15 (IL-15) and optionally chemokine (C-C motif) ligand 19 (CCL19), a pharmaceutical composition comprising immune cells, mesothelin Expression vectors comprising a polynucleotide encoding a cell surface molecule that specifically recognizes thelin, a polynucleotide encoding IL-15, and optionally a polynucleotide encoding CCL19, methods of use, and specification of human mesothelin to immune cells A cell surface molecule that specifically recognizes human mesothelin, comprising introducing a polynucleotide encoding a cell surface molecule that specifically recognizes human mesothelin, comprising the step of introducing a polynucleotide encoding a polynucleotide encoding IL-15 and optionally a polynucleotide encoding CCL19, It relates to a method of producing immune cells expressing IL-15 and optionally CCL19.

Malignant tumors are diseases that affect many people worldwide and are commonly treated with chemotherapy, radiotherapy, or surgery. However, various problems are occurring, such as the occurrence of adverse reactions, loss of some functions, and recurrence or metastasis that cannot be treated. Accordingly, the development of immune cell therapy has recently been underway to maintain a higher quality of life (QOL) of patients. Immune cell therapy involves harvesting immune cells from a patient, performing procedures to enhance the immune function of the harvested immune cells, amplifying the cells, and administering the cells back to the patient. For example, immune cell therapy involves collecting T cells from a patient, introducing a nucleic acid encoding a chimeric antigen receptor (constitutive androstane receptor, hereinafter also referred to as “CAR”) into the T cells, and This may include re-administration to the patient. Early success has been observed in hematologic malignancies using CAR-T therapy; Life-threatening toxicities and lack of substantial efficacy have also been observed in the treatment of solid tumors. Accordingly, improved CAR-T therapies are needed.

Patent Document 1: WO2020/045610 Patent Document 2: US2020/0101142 Patent Document 3: WO2013/063419

Immunotherapy using T cells presents a number of problems, including insufficient trafficking to solid tumors, high toxicity to normal tissues, inability to overcome the immunosuppressive tumor microenvironment, and insufficient activation of endogenous immune responses. do. Additionally, immune cells modified to express CARs that specifically recognize mesothelin have been shown to show minimal therapeutic efficacy. Therefore, an objective technical challenge to be solved is to provide optimized modified immune cells that target mesothelin-expressing cancers.

We have discovered that immune cells modified to express CAR, IL-15 and optionally CCL19 that specifically recognize mesothelin can improve the therapeutic efficacy of immunotherapy and improve survival.

A first polynucleotide encoding a chimeric antigen receptor (CAR) comprising an antibody specifically recognizing human mesothelin and a 4-1BB intracellular region, and a second polynucleotide encoding interleukin 15 (IL-15) An isolated nucleic acid molecule, comprising: In some embodiments, the CAR further comprises a CD8 hinge region, a CD8 transmembrane region, and a CD3ζ intracellular region. In some embodiments, the isolated nucleic acid molecule comprises a first polynucleotide encoding CAR and a second polynucleotide encoding IL-15. In some embodiments, the isolated nucleic acid molecule further comprises a third polynucleotide encoding CCL19. In some embodiments, IL-15 is human IL-15. In some embodiments, CCL19 is human CCL19. In some embodiments, the antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein VH comprises three complementarity-determining regions (CDRs) comprising SEQ ID NOs: 16-18, and VL is It contains 3 CDRs containing SEQ ID NOs: 19-21. In some embodiments, the VH comprises SEQ ID NO:22 and the VL comprises SEQ ID NO:23. In some embodiments, the antibody comprises a single-chain variable fragment (scFv) format. In some embodiments, the antibody comprises SEQ ID NO:1. In some embodiments, the 4-1BB intracellular region comprises SEQ ID NO:24. In some embodiments, the CD3ζ intracellular region comprises SEQ ID NO:25. In some embodiments, the 4-1BB intracellular region is upstream of the CD3ζ intracellular region in the isolated nucleic acid molecule. In some embodiments, the CD8 hinge region comprises SEQ ID NO:26. In some embodiments, the CD8 transmembrane region comprises SEQ ID NO:27. In some embodiments, the CAR further comprises a peptide linker of 3 to 10 amino acid residues in length connecting the antibody and the CD8 hinge region. In some embodiments, the peptide linker comprises SEQ ID NO:4. In some embodiments, the CAR molecule further comprises a signaling peptide. In some embodiments, the signaling peptide is located upstream of an antibody that specifically recognizes human mesothelin in an isolated nucleic acid molecule. In some embodiments, the signaling peptide comprises SEQ ID NO:2. In some embodiments, the second polynucleotide encoding IL-15 and optionally the third polynucleotide encoding CCL19 are each independently transcribed under a promoter comprising a polynucleotide encoding a self-cleaving 2A peptide (2A peptide). . In some embodiments, the 2A peptide comprises SEQ ID NO:5. In some embodiments, IL-15 comprises a sequence selected from SEQ ID NOs: 8-11. In some embodiments, IL-15 comprises a sequence selected from SEQ ID NO: 28 or 29. In some embodiments, CCL19 comprises SEQ ID NO:13. In some embodiments, the first polynucleotide encoding CAR and the second polynucleotide encoding IL-15 are arranged in a nucleic acid molecule from 5' end to 3' end, the first polynucleotide encoding CAR - IL-15. It is arranged like the second polynucleotide that encodes it. In some embodiments, the first polynucleotide encoding CAR, the second polynucleotide encoding IL-15, and the third polynucleotide encoding CCL19, from the 5' end to the 3' end in the nucleic acid molecule, encode the CAR. A first polynucleotide encoding - a second polynucleotide encoding IL-15 - a third polynucleotide encoding CCL19. In some embodiments, the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO: 14 or 31. In some embodiments, the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO: 34, 35, 36, or 15. In some embodiments, the isolated nucleic acid molecule comprises SEQ ID NO: 37, 38, 39, or 40.

In certain embodiments, vectors comprising nucleic acid molecules described herein are disclosed herein. In some embodiments, the vector is a viral vector, optionally an expression vector. In some embodiments, the viral vector is selected from retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated virus (AAV) vectors. In some embodiments, the viral vector is a pSFG vector, pMSGV vector, or pMSCV vector. In some embodiments, the vector is a plasmid.

In certain embodiments, disclosed herein are immune cells comprising nucleic acid molecules described herein or vectors described herein. In some embodiments, the immune cell further comprises a polynucleotide encoding gamma-TCR (γTCR) and a polynucleotide encoding delta-TCR (δTCR). In some embodiments, the γTCR is V gamma 9 TCR (Vγ9 TCR) and the δTCR is V delta 2 TCR (Vδ2TCR).

In certain embodiments, disclosed herein are immune cells that express: a) an antibody that specifically recognizes human mesothelin, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular region, and a CD3ζ intracellular region. Chimeric antigen receptor (CAR) containing; and b) IL-15; and c) optionally CCL19. In some embodiments, the immune cell is a T cell, natural killer (NK) cell, B cell, antigen presenting cell, or granulocyte, optionally a T cell or NK cell. In some embodiments, the immune cells are derived from induced pluripotent stem cells (iPSCs). In some embodiments, the immune cells do not express alpha TCR (αTCR) and/or beta TCR (βTCR), optionally αβTCR. In some embodiments, the immune cells express γδTCR. In some embodiments, the γδTCR includes Vγ9 TCR and Vδ2TCR.

In certain embodiments, disclosed herein are pharmaceutical compositions comprising an immune cell described herein and a pharmaceutically acceptable excipient.

In certain embodiments, disclosed herein is a method of treating a mesothelin-expressing cancer comprising administering an immune cell described herein or a pharmaceutical composition described herein to a subject in need of treatment of the mesothelin-expressing cancer. do. In some embodiments, the mesothelin-expressing cancer is optionally mesothelioma, colorectal cancer, pancreatic cancer, thymic cancer, bile duct cancer, lung cancer, skin cancer, breast cancer, prostate cancer, urinary bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, spleen cancer, It is a solid tumor selected from tracheal cancer, bronchial cancer, stomach cancer, esophageal cancer, gallbladder cancer, testicular cancer, ovarian cancer, and bone cancer. In some embodiments, the mesothelin-expressing cancer is a hematopoietic cancer. In some embodiments, the mesothelin-expressing cancer is a sarcoma, optionally selected from chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma. In some embodiments, the mesothelin-expressing cancer is a metastatic cancer. In some embodiments, the mesothelin-expressing cancer is a relapsed cancer or a refractory cancer. In some embodiments, the method further comprises administering an additional therapeutic agent or additional therapeutic regimen to the subject. In some embodiments, the additional therapeutic agent includes a chemotherapy agent, an immunotherapy agent, a targeted therapy, radiation therapy, or a combination thereof. In some embodiments, the additional therapeutic regimen includes first line therapy. In some embodiments, additional therapeutic regimens include surgery. In some embodiments, the immune cells described herein or the pharmaceutical composition described herein and the additional therapeutic agent are administered simultaneously. In some embodiments, the immune cells described herein or the pharmaceutical composition described herein and the additional therapeutic agent are administered sequentially. In some embodiments, the immune cells described herein or the pharmaceutical compositions described herein are administered to the subject prior to administration of additional therapeutic agents. In some embodiments, the immune cells described herein or the pharmaceutical compositions described herein are administered to the subject following administration of an additional therapeutic agent. In some embodiments, the subject is a human.

In certain embodiments, disclosed herein are methods of reducing tumor cell proliferation, comprising contacting the tumor cells with an immune cell described above to reduce tumor cell proliferation. In some embodiments, the method is an in vitro method. In some embodiments, the method is an in vivo method.

In certain embodiments, disclosed herein are methods of producing immune cells expressing a cell surface molecule that specifically recognizes human mesothelin, IL-15, and optionally CCL19, comprising: Inducing the expression of IL-15, and optionally CCL19, a cell surface molecule that specifically recognizes human mesothelin by immune cells, by introducing the nucleic acid molecule described above or the vector described above. In some embodiments, the immune cell is a T cell, natural killer (NK) cell, B cell, antigen presenting cell, or granulocyte, optionally a T cell or NK cell.

In certain embodiments, a nucleic acid molecule described above; Disclosed herein are kits comprising the vector described above, the immune cell described above, or the pharmaceutical composition described above, and instructions for use.

The immune cells of the invention have cytotoxic activity against cancer cells expressing mesothelin (eg, human mesothelin) and can suppress the formation of tumors expressing mesothelin (eg, human mesothelin). Additionally, the immune cells of the present invention have a suppressive effect on recurring cancer cells.

Figure 1A depicts a cartoon representation of an exemplary vector comprising a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically recognizes mesothelin and a polynucleotide encoding IL-15.
Figure 1B shows a cartoon representation of an exemplary vector comprising a polynucleotide encoding a chimeric antigen receptor (CAR) that specifically recognizes mesothelin, a polynucleotide encoding IL-15, and a polynucleotide encoding CCL19. do.
Figure 1C depicts a cartoon representation of modified immune cells expressing CAR and IL-15 that specifically recognize mesothelin.
Figure 1D depicts a cartoon representation of modified immune cells expressing CAR, IL-15 and CCL19 that specifically recognize mesothelin.
Figure 2 shows the results obtained by staining the obtained cells using a set of antibodies (Vδ1 Myltenyi FITC, Vδ2 Miltenyi APC, γδTCR BD BV510, CD3 BioLegend APC/Cy7 and αβTCR eBioscience FITC). Filled peaks show the results of the non-staining group, and empty peaks show the results of staining using the respective antigen-specific antibodies.
Figure 3 shows the results of flow cytometry obtained by staining the obtained cells using a set of antibodies (Vδ1 Miltenyi FITC, Vδ2 Miltenyi APC, γδTCR BD BV510, CD3 BioLegend APC/Cy7 and αβTCR eBioscience FITC).
Figure 4 shows the results of measurement of cell proliferation of iPS cell-derived γδT cells (iγδT cells). The vertical axis shows the cell proliferation rate and the horizontal axis shows the number of days since stimulation with anti-CD3 antibody (UCHT1) and anti-CD30 antibody was started.
Figure 5 shows the expression of CD3 and γδTCR molecules on the cell membrane surface of γδT cells (iγ9δ2T cells) differentiated by introducing the Vγ9Vδ2TCR gene into iPS cells.
Figure 6 shows the measurement results of cell proliferation of imesothelin-CAR/IL-15γ9δ2T cells. The vertical axis shows the number of cells, and the horizontal axis shows the number of days since stimulation with anti-CD3 antibody (UCHT1) was started.
Figure 7A depicts mesothelin staining for tumor cell lines.
Figure 7B shows that iMeso-CAR/IL-15γ9δ2T cells kill mesothelin positive cell lines in vitro in a dose-dependent manner.
Figure 8 shows that iMeso-CAR/IL-15γδT cells containing costimulatory domains CD28 or 4-1BB outperform other costimulatory domains tested.
Figures 9A-9D show that iMeso-CAR/IL-15γ9δ2T cells with mIL15/Ra or sushi15 show higher anti-tumor efficacy than other iMeso-CAR/IL-15γ9δ2T cells tested. Figures 9A and 9B show Arm 1 regimen (5x10 ⁶ cells). Figures 9C and 9D show Arm 2 regimen (1.5x10 ⁶ cells).
Figure 9E shows that iMeso-CAR/IL-15γ9δ2T cells containing 4-1BB show higher anti-tumor effect than iMeso-CAR/IL-15γ9δ2T cells containing CD28.
Figure 10 depicts GSU sc xenografts using intratumoral iMeso-CAR/IL-15γ9δ2T cell injection. iMeso-CAR/IL-15γ9δ2T cells expressing mIL15/Ra or sushi15 show higher efficacy than other iMeso-CAR/IL-15γ9δ2T cells tested.
Figure 11 depicts the efficacy of iMeso-CAR/IL-15γ9δ2T cells administered ip and iv in an intraperitoneal GSU-redLuc xenograft model.
Figure 12A depicts CAR-T cells counted by flow cytometry and FACS phenotyping performed at indicated time points.
Figure 12B shows cytotoxicity in anti-Msln CAR-T cells co-expressing CAR against Msln with a TGFβ modulator (e.g., TGFβR2-VH or dnTGFbR2) or control VH against GFP (Msln-Control VH). Show percent.

engineered immune cells

In certain embodiments, disclosed herein are engineered immune cells that express engineered cell surface molecules that specifically bind mesothelin, interleukin 15 (IL-15) and, optionally, chemokine (C-C motif) ligand 19 (CCL19). . In some embodiments, the engineered cell surface molecule comprises a chimeric antigen receptor (CAR) that specifically recognizes mesothelin or a T cell receptor (TCR) that specifically binds mesothelin.

In some embodiments, the engineered immune cell contains an exogenous nucleic acid encoding the engineered cell surface molecule, an exogenous nucleic acid encoding IL-15, and optionally an exogenous nucleic acid encoding CCL19. In some embodiments, the engineered immune cells express surface molecules that specifically recognize mesothelin, IL-15 and optionally CCL19.

Mesothelin (MSLN) is a cell surface-bound glycosylphosphatidylinositol (GPI) anchored protein, where normal expression is restricted to mesothelial cells such as the pleura, pericardium, peritoneum, testicular tunica vaginalis, ovaries, or fallopian tubes. However, MSLN can also be used to treat malignant mesothelioma, ovarian cancer, breast cancer (e.g., triple-negative breast cancer, TNBC), pancreatic cancer, lung cancer, gastric cancer, endometrial cancer, cervical cancer, biliary tract cancer, uterine serous carcinoma, cholangiocarcinoma, and pediatric acute myeloid cancer. It has been shown to be overexpressed in excessive cancers such as leukemia. Additionally, increased MSLN expression is associated with poorer prognosis in patients with TNBC, ovarian cancer, lung adenocarcinoma, cholangiocarcinoma, and pancreatic adenocarcinoma.

The physiological and biological functions of MSLN have not been fully elucidated. However, MSLN has been shown to be involved in several mechanisms of cancer pathogenesis. For example, in epithelial ovarian carcinoma, patients exhibiting higher levels of MSLN mRNA expression in surgically-resected ovarian cancer tissue are platinum and It showed resistance to chemotherapy using cyclophosphamide (Tang, et al., "The role of mesothelin in tumor progression and targeted therapy," Anticancer Agents Med Chem . 13 (2): 276-280 (2013)) . MSLN has also been found to bind with high affinity to the surface mucin MUC16 (or CA125), and binding has been suggested to mediate adhesion of ovarian cancer cells to mesothelial cells and promote metastasis (Rump, et al., “Binding of ovarian cancer antigen CA125/MUC16 to mesothelin mediates cell adhesion," J Biol Chem 279 (10): 9190-9198, 2004; Gubbels, et al., "Mesothelin-MUC16 binding is a high affinity, N-glycan dependent interaction that facilitates peritoneal metastasis of ovarian tumors," Mol Cancer 5 (1): 50, 2006). Additionally, MSLN has been shown to be involved in tumor progression, cell survival and proliferation of pancreatic cancer both in vitro and in vivo (Li, et al., “Mesothelin is a malignant factor and therapeutic vaccine target for pancreatic cancer,” Mol Cancer Ther . 7 (2): 286-296, 2008).

Chimeric Antigen Receptor (CAR)

A. anti-mesothelin antibody

In some embodiments, the engineered cell surface molecule comprises a chimeric antigen receptor (CAR) comprising an antibody that specifically recognizes mesothelin. In some embodiments, the antibody specifically recognizes mammalian mesothelin, such as rodent mesothelin, non-human primate mesothelin, or human mesothelin.

Human mesothelin, a 40 kDa protein, is encoded by the MSLN gene. Sequence information for human mesothelin can be suitably obtained by searching publicly known literature or databases, such as NCBI (www.ncbi.nlm.nih.gov/guide/). Examples of amino acid sequence information for human mesothelin may include GenBank accession numbers NP_037536.2, AAV87530.1, and their isoforms.

In some embodiments, the anti-mesothelin antibody has a heavy chain variable region (VH) comprising or consisting of CDRH1 as set forth in SEQ ID NO: 16, CDRH2 as set forth in SEQ ID NO: 17, and CDRH3 as set forth in SEQ ID NO: 18; and a light chain variable region (VL) comprising or consisting of CDRL1 as set forth in SEQ ID NO: 19, CDRL2 as set forth in SEQ ID NO: 20, and CDRL3 as set forth in SEQ ID NO: 21. See Table 1.

Table 1

*Anti-mesothelin antibody is also referred to herein as P4.

In some embodiments, the anti-mesothelin antibody comprises a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:22. a heavy chain variable region (VH) comprising; and a light chain variable region (VL) comprising a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO: 23. do. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence with about 80% sequence identity to SEQ ID NO: 22; and a light chain variable region (VL) comprising a sequence with about 80% sequence identity to SEQ ID NO:23. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence with about 85% sequence identity to SEQ ID NO: 22; and a light chain variable region (VL) comprising a sequence with about 85% sequence identity to SEQ ID NO:23. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence with about 90% sequence identity to SEQ ID NO: 22; and a light chain variable region (VL) comprising a sequence with about 90% sequence identity to SEQ ID NO:23. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence with about 95% sequence identity to SEQ ID NO: 22; and a light chain variable region (VL) comprising a sequence with about 95% sequence identity to SEQ ID NO: 23. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence with about 96% sequence identity to SEQ ID NO: 22; and a light chain variable region (VL) comprising a sequence with about 96% sequence identity to SEQ ID NO: 23. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence with about 97% sequence identity to SEQ ID NO: 22; and a light chain variable region (VL) comprising a sequence with about 97% sequence identity to SEQ ID NO: 23. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence with about 98% sequence identity to SEQ ID NO: 22; and a light chain variable region (VL) comprising a sequence with about 98% sequence identity to SEQ ID NO:23. In some embodiments, the anti-mesothelin antibody comprises a heavy chain variable region (VH) comprising a sequence with about 99% sequence identity to SEQ ID NO: 22; and a light chain variable region (VL) comprising a sequence with about 99% sequence identity to SEQ ID NO: 23. The anti-mesothelin antibody may comprise a heavy chain variable region (VH) comprising SEQ ID NO:22 and a light chain variable region (VL) comprising SEQ ID NO:23. The anti-mesothelin antibody may comprise a heavy chain variable region (VH) consisting of SEQ ID NO: 22 and a light chain variable region (VL) consisting of SEQ ID NO: 23.

In some embodiments, one or more residues within the framework region are modified in the anti-mesothelin antibody, such that 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the VH or VL region. Generates sequence identity. The term 'framework region' refers to the region of an antibody excluding the complementarity-determining region (CDR). In some embodiments, the anti-mesothelin antibody comprises one or more modifications within a framework region that are 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% relative to SEQ ID NO:22. Has a sequence that contains sequence identity. In some embodiments, the anti-mesothelin antibody has a sequence comprising one or more modifications within a framework region and comprising 85% sequence identity to SEQ ID NO:22. In some embodiments, the anti-mesothelin antibody has a sequence that includes one or more modifications within a framework region and that includes 90% sequence identity to SEQ ID NO:22. In some embodiments, the anti-mesothelin antibody has a sequence that includes one or more modifications within a framework region and that includes 95% sequence identity to SEQ ID NO:22. In some embodiments, the anti-mesothelin antibody has a sequence that includes one or more modifications within a framework region and that includes 96% sequence identity to SEQ ID NO:22. In some embodiments, the anti-mesothelin antibody has a sequence that includes one or more modifications within a framework region and that includes 97% sequence identity to SEQ ID NO:22. In some embodiments, the anti-mesothelin antibody has a sequence comprising one or more modifications within a framework region and comprising 98% sequence identity to SEQ ID NO:22. In some embodiments, the anti-mesothelin antibody has a sequence that includes one or more modifications within a framework region and that includes 99% sequence identity to SEQ ID NO:22. In some embodiments, the anti-mesothelin antibody comprises one or more modifications within a framework region that are 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% relative to SEQ ID NO:23. Has a sequence that contains sequence identity. In some embodiments, the anti-mesothelin antibody has a sequence comprising one or more modifications within a framework region and comprising 85% sequence identity to SEQ ID NO:23. In some embodiments, the anti-mesothelin antibody has a sequence that includes one or more modifications within a framework region and that includes 90% sequence identity to SEQ ID NO:23. In some embodiments, the anti-mesothelin antibody has a sequence that includes one or more modifications within a framework region and that includes 95% sequence identity to SEQ ID NO:23. In some embodiments, the anti-mesothelin antibody has a sequence that includes one or more modifications within a framework region and that includes 96% sequence identity to SEQ ID NO:23. In some embodiments, the anti-mesothelin antibody has a sequence that includes one or more modifications within a framework region and that includes 97% sequence identity to SEQ ID NO:23. In some embodiments, the anti-mesothelin antibody comprises one or more modifications within the framework region and has a sequence comprising 98% sequence identity to SEQ ID NO:23. In some embodiments, the anti-mesothelin antibody has a sequence that includes one or more modifications within a framework region and that includes 99% sequence identity to SEQ ID NO:23.

In some embodiments, the anti-mesothelin antibody comprises a single-chain variable fragment (scFv) format. In some embodiments, the anti-mesothelin scFv antibody comprises a VH comprising or consisting of CDRH1 as set forth in SEQ ID NO: 16, CDRH2 as set forth in SEQ ID NO: 17, and CDRH3 as set forth in SEQ ID NO: 18; and a VL comprising or consisting of CDRL1 as set forth in SEQ ID NO: 19, CDRL2 as set forth in SEQ ID NO: 20, and CDRL3 as set forth in SEQ ID NO: 21. In some embodiments, the anti-mesothelin scFv antibody has a sequence with about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:22 VH containing; and a VL comprising a sequence having about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:23.

In some embodiments, the VH and VL of the anti-mesothelin scFv antibody are joined via a peptide linker. 3 or more peptide linkers, e.g., about 3 to about 30, about 3 to about 25, about 3 to about 20, about 4 to about 30, about 4 to about 20, about 4 to about It may comprise 10, about 5 to about 30, about 5 to about 20, or about 5 to about 10 amino acid residues. The peptide linker may contain 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 amino acid residues.

The peptide linker may comprise a plurality of poly-alanine, poly-glycine, or a mixture of alanine and glycine residues. The peptide linker may comprise a (Gly ₄ Ser)n linker, where n is 1 to 30, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, more preferably 15, 16, 17, 18, 19 or It is an integer of 20. In some cases, the peptide linker comprises GILGSGGGGSGGGGSGGGGS (SEQ ID NO: 45) and the peptide linker comprises GGGGSGGGGSGGGGS (SEQ ID NO: 30). In some cases, the peptide linker includes GSGGGGSGGGGSGGGGS (SEQ ID NO: 41). In some cases, the peptide linker includes SEQ ID NO:45.

In some embodiments, the anti-mesothelin scFv antibody is QVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRS About 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to GINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS (SEQ ID NO: 1) Sequences included, linkers are indicated in italics. The anti-mesothelin scFv is also referred to herein as P4. In some embodiments, the anti-mesothelin scFv antibody comprises a sequence comprising about 85% sequence identity to SEQ ID NO:1. In some embodiments, the anti-mesothelin scFv antibody comprises a sequence comprising about 90% sequence identity to SEQ ID NO:1. In some embodiments, the anti-mesothelin scFv antibody comprises a sequence comprising about 95% sequence identity to SEQ ID NO:1. In some embodiments, the anti-mesothelin scFv antibody comprises a sequence comprising about 96% sequence identity to SEQ ID NO:1. In some embodiments, the anti-mesothelin scFv antibody comprises a sequence comprising about 97% sequence identity to SEQ ID NO:1. In some embodiments, the anti-mesothelin scFv antibody comprises a sequence comprising about 98% sequence identity to SEQ ID NO:1. In some embodiments, the anti-mesothelin scFv antibody comprises a sequence comprising about 99% sequence identity to SEQ ID NO:1. In some embodiments, the anti-mesothelin scFv antibody comprises SEQ ID NO:1. In some embodiments, the anti-mesothelin scFv antibody consists of SEQ ID NO:1.

B. Signaling peptides for CAR

In some embodiments, the chimeric antigen receptor (CAR) disclosed herein includes a signaling peptide (e.g., as a leader sequence). Signaling peptides can localize CARs to the surface of cells. Signaling peptides include immunoglobulin heavy chain, immunoglobulin light chain, CD8, T cell receptor α and β chains, CD3ζ, CD28, CD3E, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86. , CD134, CD137, ICOS, CD154 or GITR-derived signal peptide (leader sequence).

In some embodiments, signaling peptides may include polypeptides of the immunoglobulin heavy chain. In some embodiments, the signaling peptide has a sequence comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to MDWTWRILFLVAAATGAHS (SEQ ID NO: 2) Includes. In some embodiments, the signaling peptide comprises a sequence comprising about 85% sequence identity to SEQ ID NO:2. In some embodiments, the signaling peptide comprises a sequence comprising about 90% sequence identity to SEQ ID NO:2. In some embodiments, the signaling peptide comprises a sequence comprising about 95% sequence identity to SEQ ID NO:2. In some embodiments, the signaling peptide comprises a sequence comprising about 96% sequence identity to SEQ ID NO:2. In some embodiments, the signaling peptide comprises a sequence comprising about 97% sequence identity to SEQ ID NO:2. In some embodiments, the signaling peptide comprises a sequence comprising about 98% sequence identity to SEQ ID NO:2. In some embodiments, the signaling peptide comprises a sequence comprising about 99% sequence identity to SEQ ID NO:2. In some embodiments, the signaling peptide comprises SEQ ID NO:2. In some embodiments, the signaling peptide consists of SEQ ID NO:2.

C. transmembrane region

In some embodiments, the anti-mesothelin antibody is linked to one or more transmembrane and intracellular signaling domains. The transmembrane region may be derived from natural or synthetic sources. Exemplary transmembrane regions include CD8, T cell receptor α and β chains, CD3ζ, CD28, CD3E (CD3 epsilon), CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, It may include polypeptides of the transmembrane region derived from CD134, CD137, ICOS, CD154 and GITR. In some embodiments, the transmembrane region comprises a CD8 transmembrane region (eg, a human CD8 transmembrane region).

In some embodiments, the transmembrane region is derived from CD8. In some embodiments, the transmembrane region comprises about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to IYIWAPLAGTCGVLLLSLVITLYCN (SEQ ID NO: 27). Includes sequence. In some embodiments, the transmembrane region comprises a sequence comprising about 85% sequence identity to SEQ ID NO:27. In some embodiments, the transmembrane region comprises a sequence comprising about 90% sequence identity to SEQ ID NO:27. In some embodiments, the transmembrane region comprises a sequence comprising about 95% sequence identity to SEQ ID NO:27. In some embodiments, the transmembrane region comprises a sequence comprising about 96% sequence identity to SEQ ID NO:27. In some embodiments, the transmembrane region comprises a sequence comprising about 97% sequence identity to SEQ ID NO:27. In some embodiments, the transmembrane region comprises a sequence comprising about 98% sequence identity to SEQ ID NO:27. In some embodiments, the transmembrane region comprises a sequence comprising about 99% sequence identity to SEQ ID NO:27. In some embodiments, the transmembrane region comprises SEQ ID NO:27. In some embodiments, the transmembrane region consists of SEQ ID NO:27. In some embodiments, the transmembrane region comprises a sequence comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity with IYIWAPLAGTCGVLLLSLVITLYC (SEQ ID NO: 63) Includes. In some embodiments, the transmembrane region comprises a sequence comprising about 85% sequence identity to SEQ ID NO:63. In some embodiments, the transmembrane region comprises a sequence comprising about 90% sequence identity to SEQ ID NO:63. In some embodiments, the transmembrane region comprises a sequence comprising about 95% sequence identity to SEQ ID NO:63. In some embodiments, the transmembrane region comprises a sequence comprising about 96% sequence identity to SEQ ID NO:63. In some embodiments, the transmembrane region comprises a sequence comprising about 97% sequence identity to SEQ ID NO:63. In some embodiments, the transmembrane region comprises a sequence comprising about 98% sequence identity to SEQ ID NO:63. In some embodiments, the transmembrane region comprises a sequence comprising about 99% sequence identity to SEQ ID NO:63. In some embodiments, the transmembrane region comprises SEQ ID NO:63. In some embodiments, the transmembrane region consists of SEQ ID NO:63.

D. extracellular hinge region

An extracellular hinge region comprising or consisting of an optional oligopeptide or polypeptide may be positioned between the transmembrane region and the cell surface molecule that recognizes mesothelin. Examples of the length of the extracellular hinge region may include 1 to 100 amino acid residues, preferably 10 to 70, 10 to 50, or 10 to 30 amino acid residues. Exemplary extracellular hinge regions may include hinge regions derived from CD8, CD28, and CD4, and immunoglobulin hinge regions. In some embodiments, the hinge region comprises the hinge region of human CD8.

In some embodiments, the extracellular hinge region has about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 26) The CD8 hinge region contains the sequence. In some embodiments, the CD8 hinge region comprises a sequence with about 85% sequence identity to SEQ ID NO:26. In some embodiments, the CD8 hinge region comprises a sequence with about 90% sequence identity to SEQ ID NO:26. In some embodiments, the CD8 hinge region comprises a sequence with about 95% sequence identity to SEQ ID NO:26. In some embodiments, the CD8 hinge region comprises a sequence with about 96% sequence identity to SEQ ID NO:26. In some embodiments, the CD8 hinge region comprises a sequence with about 97% sequence identity to SEQ ID NO:26. In some embodiments, the CD8 hinge region comprises a sequence with about 98% sequence identity to SEQ ID NO:26. In some embodiments, the CD8 hinge region comprises a sequence with about 99% sequence identity to SEQ ID NO:26. In some embodiments, the CD8 hinge region comprises SEQ ID NO:26. In some embodiments, the CD8 hinge region consists of SEQ ID NO:26. In some embodiments, the CD8 hinge region has a sequence with about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 64) Includes. In some embodiments, the CD8 hinge region comprises a sequence with about 85% sequence identity to SEQ ID NO:64. In some embodiments, the CD8 hinge region comprises a sequence with about 90% sequence identity to SEQ ID NO:64. In some embodiments, the CD8 hinge region comprises a sequence with about 95% sequence identity to SEQ ID NO:64. In some embodiments, the CD8 hinge region comprises a sequence with about 96% sequence identity to SEQ ID NO:64. In some embodiments, the CD8 hinge region comprises a sequence with about 97% sequence identity to SEQ ID NO:64. In some embodiments, the CD8 hinge region comprises a sequence with about 98% sequence identity to SEQ ID NO:64. In some embodiments, the CD8 hinge region comprises a sequence with about 99% sequence identity to SEQ ID NO:64. In some embodiments, the CD8 hinge region comprises SEQ ID NO:64. In some embodiments, the CD8 hinge region consists of SEQ ID NO:64.

In some embodiments, the anti-mesothelin scFv antibody is linked to the hinge region via a peptide linker. The peptide linkers may be three or more, for example, about 3 to about 30, about 3 to about 20, 3 to about 10, about 4 to about 30, about 4 to about 20, about 4 to about 10. , about 5 to about 30, about 5 to about 20, or about 5 to about 10 amino acid residues. The peptide linker may contain 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 amino acid residues.

The peptide linker may comprise a plurality of poly-alanine, poly-glycine, a mixture of alanine and glycine residues, or a mixture of alanine or glycine and one or more additional amino acids. In some embodiments, the peptide linker comprises ArgAlaAlaAla (“RAAA”) (SEQ ID NO: 4). In some embodiments, the peptide linker is ArgAlaAlaAla (“RAAA”) (SEQ ID NO: 4). In some embodiments, the peptide linker comprises AlaAlaAla (“AAA”). In some embodiments, the peptide linker is a triple alanine linker or AlaAlaAla (“AAA”).

In some embodiments, the anti-mesothelin scFv antibody is linked to the hinge region without a linker.

E. Intracellular signaling domain (co-stimulatory domain)

In some embodiments, the CAR comprises one or more intracellular signaling regions. The intracellular signaling region (note that the intracellular signaling region is also referred to herein as the “co-stimulatory domain”) may comprise a region that can transmit a signal into the cell when a cell surface molecule recognizes mesothelin. The intracellular signaling region may comprise at least one or more members selected from the intracellular region of CD28, 4-1BB (CD137), GITR, CD27, OX40, HVEM, CD3ζ or an Fc receptor-associated polypeptide of the γ chain. In some embodiments, the intracellular signaling region comprises a polypeptide from the CD28 intracellular region (costimulatory domain), a polypeptide from the 4-1BB intracellular region (costimulatory domain), a polypeptide from the CD3 intracellular region, or a combination thereof. .

In some embodiments, the CAR comprises a 4-1BB intracellular region (costimulatory domain). In some embodiments, the 4-1BB intracellular region (co-stimulatory domain) is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% for KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL (SEQ ID NO: 24) or a sequence comprising 100% sequence identity. In some embodiments, the 4-1BB intracellular region comprises a sequence comprising about 85% sequence identity to SEQ ID NO:24. In some embodiments, the 4-1BB intracellular region comprises a sequence comprising about 90% sequence identity to SEQ ID NO:24. In some embodiments, the 4-1BB intracellular region comprises a sequence comprising about 95% sequence identity to SEQ ID NO:24. In some embodiments, the 4-1BB intracellular region comprises a sequence comprising about 96% sequence identity to SEQ ID NO:24. In some embodiments, the 4-1BB intracellular region comprises a sequence comprising about 97% sequence identity to SEQ ID NO:24. In some embodiments, the 4-1BB intracellular region comprises a sequence comprising about 98% sequence identity to SEQ ID NO:24. In some embodiments, the 4-1BB intracellular region comprises a sequence comprising about 99% sequence identity to SEQ ID NO:24. In some embodiments, the 4-1BB intracellular region comprises SEQ ID NO:24. In some embodiments, the 4-1BB intracellular region consists of SEQ ID NO:24.

In some embodiments, the CAR further comprises a CD3ζ intracellular region. In some embodiments, the CD3ζ intracellular region is about 80%, 85%, 90%, 95%, 96%, 97% for RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (SEQ ID NO: 25) , contains 98%, 99%, or 100% sequence identity. Contains a sequence that does. In some embodiments, the CD3ζ intracellular region comprises a sequence comprising about 85% sequence identity to SEQ ID NO:25. In some embodiments, the CD3ζ intracellular region comprises a sequence comprising about 90% sequence identity to SEQ ID NO:25. In some embodiments, the CD3ζ intracellular region comprises a sequence comprising about 95% sequence identity to SEQ ID NO:25. In some embodiments, the CD3ζ intracellular region comprises a sequence comprising about 96% sequence identity to SEQ ID NO:25. In some embodiments, the CD3ζ intracellular region comprises a sequence comprising about 97% sequence identity to SEQ ID NO:25. In some embodiments, the CD3ζ intracellular region comprises a sequence comprising about 98% sequence identity to SEQ ID NO:25. In some embodiments, the CD3ζ intracellular region comprises a sequence comprising about 99% sequence identity to SEQ ID NO:25. In some embodiments, the CD3ζ intracellular region comprises SEQ ID NO:25. In some embodiments, the CD3ζ intracellular region consists of SEQ ID NO:25.

IL-15

Interleukin 15 (IL-15) is involved in lymphocyte development, differentiation and homeostasis. IL-15 also stimulates CD8 T cells and induces natural killer (NK) cell activation. Within the tumor microenvironment, expression of IL-15 correlates with enhanced antitumor response. Furthermore, expression of IL-15 has shown benefit during ex vivo expansion of T cells and improves the efficacy of CAR-T cells.

As used herein, the term “IL-15” includes full-length IL-15 protein as well as fragments, as long as the function of IL-15 in the effect of the present invention is maintained. Additionally, the term “IL-15” includes wild-type IL-15 and variants thereof, including one or more mutations. In some cases, mutations include deletions, substitutions and/or additions. For example, IL-15 may contain one or more substitutions. IL-15 may also contain truncations of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or more amino acid residues, where the truncations are at the N-terminus of IL-15 , may be located at the C-terminus or in the internal region. Additionally, IL-15 or variants thereof can be linked to other proteins to form fusion proteins (e.g., linked to all or part of IL-15Rα). IL-15 may be a membrane border protein or a secreted protein.

A. mIL15/Rα

In some cases, IL-15 includes membrane bound IL-15 fused to the IL-15 receptor α chain (IL-15Rα). The fusion IL-15 protein is referred to herein as mIL15/Ra. IL-15Rα includes the entire wild-type IL-15Rα (without signal peptide) as well as functional fragments thereof (e.g., the extracellular domain or sushi domain of IL-15Rα) and IL-15Rα, optionally containing one or more substitutions, deletions or additions. Also includes variants of 15Rα.

In some cases, the mIL15/Ra fusion protein comprises an IL-15Ra polypeptide fused to the C-terminus of a membrane bound IL-15 polypeptide, either directly or indirectly through a linker. One or more peptide linkers, e.g., about 1 to about 30, about 3 to about 20, 3 to about 10, about 4 to about 30, about 4 to about 20, about 4 to about 10. , about 5 to about 30, about 5 to about 20, or about 5 to about 10 amino acid residues. The peptide linker may contain 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 amino acid residues.

The peptide linker may comprise a plurality of poly-alanine, poly-glycine, or a mixture of alanine and glycine residues. The peptide linker may comprise a (Gly ₄ Ser)n linker, where n is 1 to 30, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, more preferably 15, 16, 17, 18, 19, It is an integer of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. In some cases, the peptide linker includes SGGGSGGGGSGGGGSGGGGSGGGSLQ (SEQ ID NO: 32). In some cases, the peptide linker includes SGGSGGGGSGGGSGGGGSLQ (SEQ ID NO: 33). In some cases, the peptide linker includes GGGGSGGGGSGGGGS (SEQ ID NO: 30). In some cases, the peptide linker includes GSGGGGSGGGGSGGGGS (SEQ ID NO: 41). In some cases, the peptide linker includes SEQ ID NO:45.

In some embodiments, the immune cells described herein express mIL15/Ra. In some embodiments, mIL15/Ra is Approximately 80%, 85%, 90%, 9 for HWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (SEQ ID NO: 8) 5%, 96%, 97%, 98%, 99%, or 100% sequence identity Contains a sequence containing. In some embodiments, mIL15/Ra comprises a sequence comprising about 85% sequence identity to SEQ ID NO:8. In some embodiments, mIL15/Ra comprises a sequence comprising about 90% sequence identity to SEQ ID NO:8. In some embodiments, mIL15/Ra comprises a sequence comprising about 95% sequence identity to SEQ ID NO:8. In some embodiments, mIL15/Ra comprises a sequence comprising about 96% sequence identity to SEQ ID NO:8. In some embodiments, mIL15/Ra comprises a sequence comprising about 97% sequence identity to SEQ ID NO:8. In some embodiments, mIL15/Ra comprises a sequence comprising about 98% sequence identity to SEQ ID NO:8. In some embodiments, mIL15/Ra comprises a sequence comprising about 99% sequence identity to SEQ ID NO:8. In some embodiments, mIL15/Ra comprises SEQ ID NO:8. In some embodiments, mIL15/Ra consists of SEQ ID NO:8.

In some cases, mIL15/Ra further comprises IL-2 signal peptide (IL2sp). In some cases, IL2sp includes MYRMQLLSCIALSLALVTNS (SEQ ID NO: 6). In some cases, IL2sp consists of SEQ ID NO:6.

일부 경우에, 본원에 기재된 면역 세포는 MYRMQLLSCIALSLALVTNSATSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SGGGSGGGGSGGGGSGGGGSGGGSLQ ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (서열번호 28)에 대해 약 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 또는 100% 서열 동일성 Expresses IL2sp-mIL15/Ra polypeptide containing. In some cases, the IL2sp-mIL15/Ra polypeptide comprises approximately 85% sequence identity to SEQ ID NO:28. In some cases, the IL2sp-mIL15/Ra polypeptide comprises approximately 90% sequence identity to SEQ ID NO:28. In some cases, the IL2sp-mIL15/Ra polypeptide comprises approximately 95% sequence identity to SEQ ID NO:28. In some cases, the IL2sp-mIL15/Ra polypeptide comprises approximately 96% sequence identity to SEQ ID NO:28. In some cases, the IL2sp-mIL15/Ra polypeptide comprises approximately 97% sequence identity to SEQ ID NO:28. In some cases, the IL2sp-mIL15/Ra polypeptide comprises approximately 98% sequence identity to SEQ ID NO:28. In some cases, the IL2sp-mIL15/Ra polypeptide comprises approximately 99% sequence identity to SEQ ID NO:28. In some cases, the IL2sp-mIL15/Ra polypeptide comprises SEQ ID NO:28. In some cases, the IL2sp-mIL15/Ra polypeptide consists of SEQ ID NO:28.

B.sushi15

In some cases, IL-15 comprises an IL-15 receptor α chain (IL-15Rα) sushi domain fused to IL-15. This fusion IL-15 protein is referred to herein as sushi15. The IL-15Rα sushi domain is fused to the C-terminus of the IL-15 polypeptide either directly or indirectly through a linker.

In some cases, the sushi domain of IL-15Ra is fused to the IL-15 polypeptide indirectly through a linker. One or more peptide linkers, e.g., about 1 to about 30, about 3 to about 20, 3 to about 10, about 4 to about 30, about 4 to about 20, about 4 to about 10. , about 5 to about 30, about 5 to about 20, or about 5 to about 10 amino acid residues. The peptide linker may contain 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30 amino acid residues.

The peptide linker may comprise a plurality of poly-alanine, poly-glycine, or a mixture of alanine and glycine residues. The peptide linker may comprise a (Gly ₄ Ser)n linker, where n is 1 to 30, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, more preferably 15, 16, 17, 18, 19, It is an integer of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. In some cases, the peptide linker includes SEQ ID NO:32. In some cases, the peptide linker includes SEQ ID NO:33. In some cases, the peptide linker includes SEQ ID NO:30. In some cases, the peptide linker includes SEQ ID NO:41. In some cases, the peptide linker includes SEQ ID NO:45.

In some embodiments, the immune cells described herein express sushi15. In some embodiments, sushi15 is ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR SGGGSGGGGSGGGGSGGGGSGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFV Comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to HIVQMFINTS (SEQ ID NO: 10) Includes sequence. In some cases, sushi15 includes a sequence comprising approximately 85% sequence identity to SEQ ID NO:10. In some cases, sushi15 includes a sequence comprising approximately 90% sequence identity to SEQ ID NO:10. In some cases, sushi15 includes a sequence comprising approximately 95% sequence identity to SEQ ID NO:10. In some cases, sushi15 includes a sequence comprising approximately 96% sequence identity to SEQ ID NO:10. In some cases, sushi15 includes a sequence comprising approximately 97% sequence identity to SEQ ID NO:10. In some cases, sushi15 includes a sequence comprising approximately 98% sequence identity to SEQ ID NO:10. In some cases, sushi15 includes a sequence comprising approximately 99% sequence identity to SEQ ID NO:10. In some cases, sushi15 includes SEQ ID NO: 10. In some cases, sushi15 consists of SEQ ID NO: 10.

In some cases, sushi15 additionally contains the IL-15Ra signal peptide (IL15Rasp). In some cases, IL15Rasp includes MAPRRARGCRTLGLPALLLLLLLRPPATRG (SEQ ID NO: 7). In some cases, IL15Rasp consists of SEQ ID NO:7.

In some instances , the immune cells described herein may be About 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to NGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 29) Expresses the IL15Rasp-sushi15 polypeptide containing. In some cases, the IL15Rasp-sushi15 polypeptide comprises approximately 85% sequence identity to SEQ ID NO:29. In some cases, the IL15Rasp-sushi15 polypeptide comprises approximately 90% sequence identity to SEQ ID NO:29. In some cases, the IL15Rasp-sushi15 polypeptide comprises approximately 95% sequence identity to SEQ ID NO:29. In some cases, the IL15Rasp-sushi15 polypeptide comprises approximately 96% sequence identity to SEQ ID NO:29. In some cases, the IL15Rasp-sushi15 polypeptide comprises approximately 97% sequence identity to SEQ ID NO:29. In some cases, the IL15Rasp-sushi15 polypeptide comprises approximately 98% sequence identity to SEQ ID NO:29. In some cases, the IL15Rasp-sushi15 polypeptide comprises approximately 99% sequence identity to SEQ ID NO:29. In some cases, the IL15Rasp-sushi15 polypeptide comprises SEQ ID NO:29. In some cases, the IL15Rasp-sushi15 polypeptide consists of SEQ ID NO:29.

C. mIL15/Ra-LSP

In some cases, IL-15 includes membrane-bound IL-15 fused to the IL-15 receptor α chain (IL-15Rα), the IL15 propeptide, and the long signal peptide (LSP) fusion protein of IL-15. This fusion IL-15 protein is referred to herein as mIL15/Ra-LSP. IL-15Rα polypeptides include the full-length IL-15Rα chain without the signal peptide. IL-15Ra is fused to the C-terminus of the IL-15 polypeptide either directly or indirectly through a linker. The IL15 propeptide is fused directly or indirectly to the C-terminus of LSP. The IL15 propeptide is fused directly or indirectly to the C-terminus of the IL-15Ra polypeptide. In some cases, the IL15 propeptide includes GIHVFILGCFSAGLPKTEA (SEQ ID NO: 48). In some cases, the IL15 propeptide consists of SEQ ID NO:48. In some cases, the LSP includes MRISKPHLRSISIQCYLCLLLLNSHFLTEA (SEQ ID NO: 47). In some cases, the LSP consists of SEQ ID NO:47.

In some cases, IL-15Ra is fused to the C-terminus of the IL-15 polypeptide indirectly through a linker. One or more peptide linkers, e.g., about 1 to about 30, about 3 to about 20, 3 to about 10, about 4 to about 30, about 4 to about 20, about 4 to about 10. , about 5 to about 30, about 5 to about 20, or about 5 to about 10 amino acid residues. The peptide linker may contain 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25 or 30 amino acid residues.

The peptide linker may comprise a plurality of poly-serine, poly-glycine, or a mixture of serine and glycine residues. The peptide linker may comprise a (Gly ₄ Ser)n linker, where n is 1 to 30, preferably 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, more preferably 15, 16, 17, 18, 19, It is an integer of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30. In some cases, the peptide linker includes SEQ ID NO:32. In some cases, the peptide linker includes SEQ ID NO:33. In some cases, the peptide linker includes SEQ ID NO:30. In some cases, the peptide linker includes SEQ ID NO:41. In some cases, the peptide linker includes SEQ ID NO:45.

In some embodiments, the immune cells described herein express mIL15/Ra-LSP. In some embodiments, mIL15/Ra-LSP is MRISKPHLRSISIQCYLCLLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPP MSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL Approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to (SEQ ID NO: 9) Contains a sequence containing. In some cases, mIL15/Ra-LSP comprises a sequence comprising approximately 85% sequence identity to SEQ ID NO:9. In some cases, mIL15/Ra-LSP comprises a sequence comprising approximately 90% sequence identity to SEQ ID NO:9. In some cases, mIL15/Ra-LSP comprises a sequence comprising approximately 95% sequence identity to SEQ ID NO:9. In some cases, mIL15/Ra-LSP comprises a sequence comprising approximately 96% sequence identity to SEQ ID NO:9. In some cases, mIL15/Ra-LSP comprises a sequence comprising approximately 97% sequence identity to SEQ ID NO:9. In some cases, mIL15/Ra-LSP comprises a sequence comprising approximately 98% sequence identity to SEQ ID NO:9. In some cases, mIL15/Ra-LSP comprises a sequence comprising approximately 99% sequence identity to SEQ ID NO:9. In some cases, mIL15/Ra-LSP includes SEQ ID NO:9. In some cases, mIL15/Ra-LSP consists of SEQ ID NO:9.

In some cases, mIL15/Ra-LSP further comprises IL-2 signal peptide (IL2sp). In some cases, IL2sp includes SEQ ID NO:6. In some cases, IL2sp consists of SEQ ID NO:6.

D.sIL15-LSP

In some embodiments, IL-15 comprises an IL-15 polypeptide, an IL-15 propeptide, and a long signal peptide (LSP) fusion protein of IL-15. This fusion IL-15 protein is referred to herein as sIL15. The IL15 propeptide is fused directly or indirectly to the C-terminus of LSP. The IL-15 polypeptide is fused directly or indirectly to the C-terminus of the IL-15 propeptide. In some cases, the IL15 propeptide includes SEQ ID NO:48. In some cases, the IL15 propeptide consists of SEQ ID NO:48. In some cases, the LSP includes SEQ ID NO:47. In some cases, the LSP consists of SEQ ID NO:47. In some cases, the immune cells described herein express sIL15. In some embodiments, sIL15 is about 80%, 85% for MRISKPHLRSISIQCYLCLLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 11) Sequences containing 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity Includes. In some cases, sIL15 includes a sequence comprising approximately 85% sequence identity to SEQ ID NO:11. In some cases, sIL15 includes a sequence comprising approximately 90% sequence identity to SEQ ID NO:11. In some cases, sIL15 includes a sequence comprising approximately 95% sequence identity to SEQ ID NO:11. In some cases, sIL15 includes a sequence comprising approximately 96% sequence identity to SEQ ID NO:11. In some cases, sIL15 includes a sequence comprising approximately 97% sequence identity to SEQ ID NO:11. In some cases, sIL15 includes a sequence comprising approximately 98% sequence identity to SEQ ID NO:11. In some cases, sIL15 includes a sequence comprising approximately 99% sequence identity to SEQ ID NO:11. In some cases, sIL15 includes SEQ ID NO: 11. In some cases, sIL15 consists of SEQ ID NO: 11.

CCL19

Chemokine (C-C motif) ligand 19 (CCL19), also known as EBl1 ligand chemokine (ELC) and macrophage inflammatory protein-3-beta (MIP-3-beta), plays a role in lymphocyte recycling and homing. CCL19 is expressed by dendritic cells or macrophages in lymph nodes and has the function of initiating the migration of T cells, B cells, or mature dendritic cells through the receptor CCR7.

In certain embodiments, the immune cells described herein further express CCL19. In some embodiments, CCL19 comprises a sequence comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to GTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSS (SEQ ID NO: 13) Includes. In some embodiments, CCL19 comprises a sequence comprising about 85% sequence identity to SEQ ID NO:13. In some embodiments, CCL19 comprises a sequence comprising about 90% sequence identity to SEQ ID NO:13. In some embodiments, CCL19 comprises a sequence comprising about 95% sequence identity to SEQ ID NO:13. In some embodiments, CCL19 comprises a sequence comprising about 96% sequence identity to SEQ ID NO:13. In some embodiments, CCL19 comprises a sequence comprising about 97% sequence identity to SEQ ID NO:13. In some embodiments, CCL19 comprises a sequence comprising about 98% sequence identity to SEQ ID NO:13. In some embodiments, CCL19 comprises a sequence comprising about 99% sequence identity to SEQ ID NO:13. In some embodiments, CCL19 comprises SEQ ID NO:13. In some embodiments, CCL19 consists of SEQ ID NO:13.

In some embodiments, CCL19 further comprises a signal peptide, MALLLASLLVLWTSPAPPTLS (SEQ ID NO: 12) (also referred to herein as endosp). In some cases, the immune cells described herein have about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence for signal peptides SEQ ID NO: 12 and SEQ ID NO: 13. Both express CCL19 containing sequences that contain identity. In some cases, CCL19 is fused to the C-terminus of the endosp.

Additional immune function control factors

Immune cells of the invention may contain additional immune function control factors, such as CCL21, IL-2, IL-4, IL-7, IL-12, IL-13, IL-17, IL-18, IP-10, interferon- γ, MIP-1alpha, GM-CSF, M-CSF, TGF-beta, XCL1, FLT3L or TNF-alpha may be additionally expressed. In some embodiments, the additional immune function control factor includes IL-2. In some embodiments, the additional immune function control factor includes IL-7. In some embodiments, the additional immune function control factor includes interferon-γ. In some embodiments, the additional immune function control factor includes GM-CSF. In some embodiments, the additional immune function control factor includes TGF-beta. In some embodiments, the additional immune function control factor includes TNF-alpha. In some embodiments, the additional immune function control factor is preferably an immune function control factor other than IL-12.

Arrangement of each area

In certain embodiments, an isolated cell comprising an engineered cell surface molecule that specifically binds mesothelin (e.g., a CAR that specifically binds mesothelin), IL-15, and optionally one or more polynucleotides encoding CCL19. Nucleic acid molecules are disclosed herein. In some embodiments, the isolated nucleic acid molecule is an antibody that specifically recognizes human mesothelin, a first antibody encoding a CAR comprising a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular region, and a CD3ζ intracellular region. polynucleotide; and a second polynucleotide encoding IL-15. In some cases, the isolated nucleic acid molecule is an antibody that specifically recognizes human mesothelin, a first poly nucleotide; a second polynucleotide encoding IL-15; and a third polynucleotide encoding CCL19. In some embodiments, the polynucleotides encoding CAR, IL-15, and CCL19 are located on two or more different polynucleotides within a nucleic acid molecule. In other embodiments, the isolated nucleic acid molecule comprises a polynucleotide encoding CAR and IL-15, a polynucleotide encoding CAR and CCL19, or a polynucleotide encoding CAR, IL-15, or CCL19.

In some embodiments, the polynucleotide encoding the CAR comprises a signaling peptide upstream of an antibody that specifically recognizes human mesothelin. In some embodiments, the antibody is linked to the CD8 hinge region by a peptide linker (e.g., SEQ ID NO:41). In some embodiments, the 4-1BB intracellular region is located upstream of the CD3ζ intracellular region in the polynucleotide.

The polynucleotide is MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGI About 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or A CAR comprising a sequence with 100% sequence identity may be encoded. The CAR may comprise a sequence with about 85% sequence identity to SEQ ID NO:3. The CAR may comprise a sequence with about 90% sequence identity to SEQ ID NO:3. The CAR may comprise a sequence with about 95% sequence identity to SEQ ID NO:3. The CAR may comprise a sequence with about 96% sequence identity to SEQ ID NO:3. The CAR may comprise a sequence with about 97% sequence identity to SEQ ID NO:3. The CAR may comprise a sequence with about 98% sequence identity to SEQ ID NO:3. The CAR may comprise a sequence with about 99% sequence identity to SEQ ID NO:3. CAR may include SEQ ID NO:3. CAR may consist of SEQ ID NO: 3.

폴리뉴클레오티드는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS RAAA PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (서열번호 46)(또한 본원에서 ssVH-P4-CAR로서 지칭됨)에 대해 약 80%, 85%, 90%, 95%, 96%, 97%, 98%, A CAR may encode a CAR comprising a sequence with 99%, or 100% sequence identity. The CAR may comprise a sequence with about 85% sequence identity to SEQ ID NO:46. The CAR may comprise a sequence with about 90% sequence identity to SEQ ID NO:46. The CAR may comprise a sequence with about 95% sequence identity to SEQ ID NO:46. The CAR may comprise a sequence with about 96% sequence identity to SEQ ID NO:46. The CAR may comprise a sequence with about 97% sequence identity to SEQ ID NO:46. The CAR may comprise a sequence with about 98% sequence identity to SEQ ID NO:46. The CAR may comprise a sequence with about 99% sequence identity to SEQ ID NO:46. The CAR may include SEQ ID NO:46. CAR may consist of SEQ ID NO: 46.

폴리뉴클레오티드는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS RAAA P TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC N KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (서열번호 43)(또한 본원에서 ssVHsp-P4-CD8힌지-TM-41BB-CD3z로서 지칭됨)에 대해 약 80%, 85%, 90%, 95% , may encode a CAR comprising a sequence having 96%, 97%, 98%, 99%, or 100% sequence identity. The CAR may comprise a sequence with about 85% sequence identity to SEQ ID NO:43. The CAR may comprise a sequence with about 90% sequence identity to SEQ ID NO:43. The CAR may comprise a sequence with about 95% sequence identity to SEQ ID NO:43. The CAR may comprise a sequence with about 96% sequence identity to SEQ ID NO:43. The CAR may comprise a sequence with about 97% sequence identity to SEQ ID NO:43. The CAR may comprise a sequence with about 98% sequence identity to SEQ ID NO:43. The CAR may comprise a sequence with about 99% sequence identity to SEQ ID NO:43. The CAR may include SEQ ID NO:43. CAR may consist of SEQ ID NO: 43.

폴리뉴클레오티드에서는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS P TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC N KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (서열번호 65)에 대해 약 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 또는 100% 서열 동일성을 갖는 서열 A CAR containing can be encoded. The CAR may comprise a sequence with about 85% sequence identity to SEQ ID NO:65. The CAR may comprise a sequence with approximately 90% sequence identity to SEQ ID NO:65. The CAR may comprise a sequence with about 95% sequence identity to SEQ ID NO:65. The CAR may comprise a sequence with about 96% sequence identity to SEQ ID NO:65. The CAR may comprise a sequence with about 97% sequence identity to SEQ ID NO:65. The CAR may comprise a sequence with about 98% sequence identity to SEQ ID NO:65. The CAR may comprise a sequence with about 99% sequence identity to SEQ ID NO:65. The CAR may include SEQ ID NO:65. CAR may consist of SEQ ID NO: 65.

폴리뉴클레오티드에서는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (서열번호 66)에 대해 약 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 또는 100% 서열 동일성을 갖는 서열을 포함하는 CAR can be encoded. The CAR may comprise a sequence with about 85% sequence identity to SEQ ID NO:66. The CAR may comprise a sequence with about 90% sequence identity to SEQ ID NO:66. The CAR may comprise a sequence with about 95% sequence identity to SEQ ID NO:66. The CAR may comprise a sequence with about 96% sequence identity to SEQ ID NO:66. The CAR may comprise a sequence with about 97% sequence identity to SEQ ID NO:66. The CAR may comprise a sequence with about 98% sequence identity to SEQ ID NO:66. The CAR may comprise a sequence with about 99% sequence identity to SEQ ID NO:66. The CAR may include SEQ ID NO:66. CAR may consist of SEQ ID NO: 66.

폴리뉴클레오티드에서는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS P TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (서열번호 67)에 대해 약 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 또는 100% 서열 동일성을 갖는 서열을 포함 CAR can be encoded. The CAR may comprise a sequence with about 85% sequence identity to SEQ ID NO:67. The CAR may comprise a sequence with about 90% sequence identity to SEQ ID NO:67. The CAR may comprise a sequence with about 95% sequence identity to SEQ ID NO:67. The CAR may comprise a sequence with about 96% sequence identity to SEQ ID NO:67. The CAR may comprise a sequence with about 97% sequence identity to SEQ ID NO:67. The CAR may comprise a sequence with about 98% sequence identity to SEQ ID NO:67. The CAR may comprise a sequence with about 99% sequence identity to SEQ ID NO:67. The CAR may include SEQ ID NO:67. CAR may consist of SEQ ID NO: 67.

폴리뉴클레오티드에서는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC N KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (서열번호 68)에 대해 약 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 또는 100% 서열 동일성을 갖는 서열을 포함 CAR can be encoded. The CAR may comprise a sequence with about 85% sequence identity to SEQ ID NO:68. The CAR may comprise a sequence with about 90% sequence identity to SEQ ID NO:68. The CAR may comprise a sequence with about 95% sequence identity to SEQ ID NO:68. The CAR may comprise a sequence with about 96% sequence identity to SEQ ID NO:68. The CAR may comprise a sequence with about 97% sequence identity to SEQ ID NO:68. The CAR may comprise a sequence with about 98% sequence identity to SEQ ID NO:68. The CAR may comprise a sequence with about 99% sequence identity to SEQ ID NO:68. The CAR may include SEQ ID NO:68. CAR may consist of SEQ ID NO: 68.

폴리뉴클레오티드는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS AAA P TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC N KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (서열번호 69)에 대해 약 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 또는 100% 서열 동일성을 갖는 A CAR containing a sequence may be encoded. The CAR may comprise a sequence with about 85% sequence identity to SEQ ID NO:69. The CAR may comprise a sequence with about 90% sequence identity to SEQ ID NO:69. The CAR may comprise a sequence with about 95% sequence identity to SEQ ID NO:69. The CAR may comprise a sequence with about 96% sequence identity to SEQ ID NO:69. The CAR may comprise a sequence with about 97% sequence identity to SEQ ID NO:69. The CAR may comprise a sequence with about 98% sequence identity to SEQ ID NO:69. The CAR may comprise a sequence with about 99% sequence identity to SEQ ID NO:69. The CAR may include SEQ ID NO:69. CAR may consist of SEQ ID NO: 69.

폴리뉴클레오티드는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS AAA TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC N KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (서열번호 70)에 대해 약 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 또는 100% 서열 동일성을 갖는 서열 A CAR containing can be encoded. The CAR may comprise a sequence with about 85% sequence identity to SEQ ID NO:70. The CAR may comprise a sequence with about 90% sequence identity to SEQ ID NO:70. The CAR may comprise a sequence with about 95% sequence identity to SEQ ID NO:70. The CAR may comprise a sequence with about 96% sequence identity to SEQ ID NO:70. The CAR may comprise a sequence with about 97% sequence identity to SEQ ID NO:70. The CAR may comprise a sequence with about 98% sequence identity to SEQ ID NO:70. The CAR may comprise a sequence with about 99% sequence identity to SEQ ID NO:70. The CAR may include SEQ ID NO:70. CAR may consist of SEQ ID NO: 70.

폴리뉴클레오티드는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS AAA P TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (서열번호 71)에 대해 약 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 또는 100% 서열 동일성을 갖는 서열을 The CAR containing it can be encoded. The CAR may comprise a sequence with about 85% sequence identity to SEQ ID NO:71. The CAR may comprise a sequence with about 90% sequence identity to SEQ ID NO:71. The CAR may comprise a sequence with about 95% sequence identity to SEQ ID NO:71. The CAR may comprise a sequence with about 96% sequence identity to SEQ ID NO:71. The CAR may comprise a sequence with about 97% sequence identity to SEQ ID NO:71. The CAR may comprise a sequence with about 98% sequence identity to SEQ ID NO:71. The CAR may comprise a sequence with about 99% sequence identity to SEQ ID NO:71. The CAR may include SEQ ID NO:71. CAR may consist of SEQ ID NO: 71.

폴리뉴클레오티드는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS RAAA TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (서열번호 72)에 대해 약 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 또는 100% 서열 동일성을 갖는 서열을 포함 CAR can be encoded. The CAR may comprise a sequence with about 85% sequence identity to SEQ ID NO:72. The CAR may comprise a sequence with about 90% sequence identity to SEQ ID NO:72. The CAR may comprise a sequence with about 95% sequence identity to SEQ ID NO:72. The CAR may comprise a sequence with about 96% sequence identity to SEQ ID NO:72. The CAR may comprise a sequence with about 97% sequence identity to SEQ ID NO:72. The CAR may comprise a sequence with about 98% sequence identity to SEQ ID NO:72. The CAR may comprise a sequence with about 99% sequence identity to SEQ ID NO:72. The CAR may include SEQ ID NO:72. CAR may consist of SEQ ID NO: 72.

폴리뉴클레오티드는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS RAAA P TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (서열번호 73)에 대해 약 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 또는 100% 서열 동일성을 갖는 서열을 The CAR containing it can be encoded. The CAR may comprise a sequence with about 85% sequence identity to SEQ ID NO:73. The CAR may comprise a sequence with about 90% sequence identity to SEQ ID NO:73. The CAR may comprise a sequence with about 95% sequence identity to SEQ ID NO:73. The CAR may comprise a sequence with about 96% sequence identity to SEQ ID NO:73. The CAR may comprise a sequence with about 97% sequence identity to SEQ ID NO:73. The CAR may comprise a sequence with about 98% sequence identity to SEQ ID NO:73. The CAR may comprise a sequence with about 99% sequence identity to SEQ ID NO:73. The CAR may include SEQ ID NO:73. CAR may consist of SEQ ID NO: 73.

폴리뉴클레오티드는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS RAAA TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC N KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (서열번호 74)에 대해 약 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 또는 100% 서열 동일성을 갖는 서열 A CAR containing can be encoded. The CAR may comprise a sequence with about 85% sequence identity to SEQ ID NO:74. The CAR may comprise a sequence with about 90% sequence identity to SEQ ID NO:74. The CAR may comprise a sequence with about 95% sequence identity to SEQ ID NO:74. The CAR may comprise a sequence with about 96% sequence identity to SEQ ID NO:74. The CAR may comprise a sequence with about 97% sequence identity to SEQ ID NO:74. The CAR may comprise a sequence with about 98% sequence identity to SEQ ID NO:74. The CAR may comprise a sequence with about 99% sequence identity to SEQ ID NO:74. The CAR may include SEQ ID NO:74. CAR may consist of SEQ ID NO: 74.

폴리뉴클레오티드는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS AAA TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPR (서열번호 75)에 대해 약 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 또는 100% 서열 동일성을 갖는 서열을 포함 CAR can be encoded. The CAR may comprise a sequence with about 85% sequence identity to SEQ ID NO:75. The CAR may comprise a sequence with about 90% sequence identity to SEQ ID NO:75. The CAR may comprise a sequence with about 95% sequence identity to SEQ ID NO:75. The CAR may comprise a sequence with about 96% sequence identity to SEQ ID NO:75. The CAR may comprise a sequence with about 97% sequence identity to SEQ ID NO:75. The CAR may comprise a sequence with about 98% sequence identity to SEQ ID NO:75. The CAR may comprise a sequence with about 99% sequence identity to SEQ ID NO:75. The CAR may include SEQ ID NO:43. CAR may consist of SEQ ID NO: 75.

본원에 기재된 CAR을 인코딩하는 폴리뉴클레오티드는 ATGGACTGGACATGGCGGATACTCTTCCTCGTCGCTGCTGCAACCGGAGCCCACAGCCAGGTGCAGCTCCAGCAGTCTGGGCCAGGTTTGGTGACTCCTAGTCAGACATTGAGCTTGACTTGTGCTATCAGCGGAGACTCTGTTTCATCTAATTCTGCAACTTGGAACTGGATTCGGCAGTCCCCCAGCCGGGGGCTCGAGTGGTTGGGTCGGACCTACTATCGGAGCAAATGGTACAATGACTATGCAGTGAGCGTCAAATCAAGAATGAGCATCAATCCTGACACAAGCAAGAACCAGTTTAGCCTTCAGCTTAATAGCGTGACTCCAGAGGACACAGCTGTGTACTATTGCGCGAGAGGCATGATGACATACTATTACGGAATGGACGTGTGGGGCCAGGGAACTACTGTTACAGTGTCAAGCGGAATCCTCGGTAGCGGAGGCGGCGGTTCCGGCGGAGGGGGTAGTGGTGGCGGGGGTAGTCAACCTGTGCTGACCCAGAGCAGCTCTCTTAGTGCTAGCCCAGGTGCAAGTGCAAGTCTTACCTGTACACTGCGCTCCGGTATTAATGTGGGCCCTTACCGAATTTACTGGTACCAGCAGAAACCAGGCTCCCCTCCCCAGTATCTGCTGAACTATAAGTCTGACTCAGACAAACAGCAGGGCTCCGGTGTGCCATCCCGATTTAGTGGCTCAAAGGATGCTAGTGCAAATGCCGGTGTTCTCCTGATCAGCGGACTCAGATCAGAGGACGAAGCAGACTATTACTGTATGATTTGGCATAGCAGCGCTGCTGTCTTCGGAGGAGGGACTCAGCTCACTGTCTTGAGTCGGGCCGCTGCACCTACCACTACCCCTGCCCCTCGACCCCCTACTCCCGCCCCAACTATCGCATCCCAACCACTCTCTCTCAGACCCGAAGCCTGTAGACCCGCAGCCGGTGGCGCTGTGCATACTCGCGGACTTGATTTTGCTTGTGATATTTATATCTGGGCCCCCCTTGCCGGAACTTGTGGAGTTCTCCTGCTGTCTCTCGTAATCACCCTTTATTGTAACAAACGGGGGCGCAAAAAACTTCTTTACATTTTCAAGCAGCCCTTTATGCGGCCCGTGCAGACCACACAGGAAGAAGATGGCTGCAGCTGCAGGTTCCCAGAGGAAGAAGAGGGCGGCTGCGAGCTGCGAGTAAAGTTCAGCCGGAGCGCCGATGCACCTGCATACCAGCAGGGTCAGAACCAGCTCTACAATGAGCTGAACCTGGGCAGAAGAGAGGAATATGATGTACTCGACAAGAGAAGGGGACGCGATCCAGAGATGGGCGGCAAACCACGGCGGAAAAATCCGCAGGAGGGGCTCTATAACGAGCTCCAGAAGGACAAGATGGCAGAAGCCTACTCAGAAATTGGCATGAAAGGAGAGAGAAGGAGGGGAAAGGGCCATGATGGCCTTTACCAAGGGTTGTCTACTGCCACCAAGGATACGTACGATGCACTCCATATGCAGGCTCTTCCTCCCCGA (서열번호 50), ATGGACTGGACATGGCGGATACTCTTCCTCGTCGCTGCTGCAACCGGAGCCCACAGCCAGGTGCAGCTCCAGCAGTCTGGGCCAGGTTTGGTGACTCCTAGTCAGACATTGAGCTTGACTTGTGCTATCAGCGGAGACTCTGTTTCATCTAATTCTGCAACTTGGAACTGGATTCGGCAGTCCCCCAGCCGGGGGCTCGAGTGGTTGGGTCGGACCTACTATCGGAGCAAATGGTACAATGACTATGCAGTGAGCGTCAAATCAAGAATGAGCATCAATCCTGACACAAGCAAGAACCAGTTTAGCCTTCAGCTTAATAGCGTGACTCCAGAGGACACAGCTGTGTACTATTGCGCGAGAGGCATGATGACATACTATTACGGAATGGACGTGTGGGGCCAGGGAACTACTGTTACAGTGTCAAGCGGAATCCTCGGTAGCGGAGGCGGCGGTTCCGGCGGAGGGGGTAGTGGTGGCGGGGGTAGTCAACCTGTGCTGACCCAGAGCAGCTCTCTTAGTGCTAGCCCAGGTGCAAGTGCAAGTCTTACCTGTACACTGCGCTCCGGTATTAATGTGGGCCCTTACCGAATTTACTGGTACCAGCAGAAACCAGGATCCCCTCCCCAGTATCTGCTGAACTATAAGTCTGACTCAGACAAACAGCAGGGCTCCGGTGTGCCATCCCGATTTAGTGGCTCAAAGGATGCTAGTGCAAATGCCGGTGTTCTCCTGATCAGCGGACTCAGATCAGAGGACGAAGCAGACTATTACTGTATGATTTGGCATAGCAGCGCTGCTGTCTTCGGAGGAGGGACTCAGCTCACTGTCTTGAGTCGGGCCGCTGCACCTACCACTACCCCTGCCCCTCGACCCCCTACTCCCGCCCCAACTATCGCATCCCAACCACTCTCTCTCAGACCCGAAGCCTGTAGACCCGCAGCCGGTGGCGCTGTGCATACTCGCGGACTTGATTTTGCTTGTGATATTTATATCTGGGCCCCCCTTGCCGGAACTTGTGGAGTTCTCCTGCTGTCTCTCGTAATCACCCTTTATTGTAACAAACGGGGGCGCAAAAAACTTCTTTACATTTTCAAGCAGCCCTTTATGCGGCCCGTGCAGACCACACAGGAAGAAGATGGCTGCAGCTGCAGGTTCCCAGAGGAAGAAGAGGGCGGCTGCGAGCTGCGAGTAAAGTTCAGCCGGAGCGCCGATGCACCTGCATACCAGCAGGGTCAGAACCAGCTCTACAATGAGCTGAACCTGGGCAGAAGAGAGGAATATGATGTACTCGACAAGAGAAGGGGACGCGATCCAGAGATGGGCGGCAAACCACGGCGGAAAAATCCGCAGGAGGGGCTCTATAACGAGCTCCAGAAGGACAAGATGGCAGAAGCCTACTCAGAAATTGGCATGAAAGGAGAGAGAAGGAGGGGAAAGGGCCATGATGGCCTTTACCAAGGGTTGTCTACTGCCACCAAGGATACGTACGATGCACTCCATATGCAGGCTCTTCCTCCCCGA (서열번호 51), 또는 ATGGATTGGACCTGGCGAATACTCTTCCTCGTCGCAGCGGCCACTGGTGCCCATTCACAAGTCCAACTGCAGCAGAGCGGACCTGGCCTGGTGACACCCAGTCAGACTCTCAGCCTGACTTGTGCAATCAGCGGCGATAGTGTGTCTAGTAATTCTGCAACATGGAACTGGATCAGACAATCACCAAGTCGGGGACTGGAGTGGCTCGGTAGAACCTATTATAGGTCCAAATGGTATAACGATTATGCAGTGTCCGTGAAGTCCCGAATGTCTATCAACCCTGATACTAGTAAGAATCAATTCAGTCTGCAGCTTAACAGCGTAACCCCCGAAGATACTGCTGTGTATTACTGTGCCCGGGGTATGATGACTTACTACTACGGAATGGATGTGTGGGGGCAGGGAACAACCGTTACTGTTTCATCCGGCATTCTCGGGAGCGGAGGCGGTGGAAGCGGTGGGGGAGGGTCCGGGGGAGGAGGATCTCAGCCTGTTCTTACTCAATCTTCTTCCCTCTCCGCCTCACCCGGGGCCTCCGCCTCACTGACCTGCACTCTGCGATCAGGCATCAACGTTGGGCCTTATAGAATCTACTGGTACCAGCAAAAGCCTGGATCACCGCCCCAGTACCTGCTGAACTATAAATCAGACTCAGACAAGCAGCAGGGCTCCGGCGTGCCGAGTCGATTTAGCGGGAGCAAGGACGCGTCTGCTAATGCCGGCGTGCTTCTCATCAGCGGGCTCCGCAGTGAGGATGAGGCAGATTACTACTGCATGATTTGGCATAGCAGTGCAGCCGTATTTGGCGGAGGAACACAGCTGACTGTCCTCTCTCGCGCCGCCGCTCCGACCACCACCCCTGCACCACGCCCACCTACTCCTGCGCCAACCATTGCCAGCCAGCCTCTCTCTCTCCGACCCGAGGCCTGTAGACCTGCCGCTGGCGGTGCAGTTCATACTCGGGGTCTCGATTTCGCCTGCGACATCTACATCTGGGCACCACTGGCTGGCACTTGTGGCGTTTTGCTCCTGTCCCTGGTGATCACTCTCTACTGTAATAAGAGGGGGAGGAAGAAACTCCTGTATATTTTCAAACAACCCTTTATGCGCCCTGTCCAAACCACCCAGGAAGAAGATGGATGTAGTTGCAGATTCCCAGAAGAAGAGGAGGGTGGGTGTGAACTTAGGGTGAAGTTTAGTCGCAGTGCCGACGCTCCCGCTTACCAACAGGGTCAGAACCAACTCTACAATGAGCTGAATCTGGGGAGGCGCGAAGAATACGACGTTCTGGATAAAAGACGCGGCCGCGACCCCGAGATGGGCGGGAAACCGCGGAGAAAGAACCCACAGGAAGGATTGTACAATGAGCTCCAGAAAGATAAGATGGCAGAAGCCTACTCCGAGATCGGCATGAAGGGGGAGCGAAGGCGCGGGAAAGGACACGATGGGCTGTACCAGGGTCTTTCAACCGCGACAAAGGACACCTATGATGCTCTCCATATGCAGGCCCTCCCGCCACGC (서열번호 52)에 대해 약 80%, 85%, 90%, 95%, 96%, 97 %, 98%, 99%, or 100% sequence identity. The polynucleotide may comprise a nucleic acid sequence having about 85% sequence identity to SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52. The polynucleotide may comprise a nucleic acid sequence having about 90% sequence identity to SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52. The polynucleotide may comprise a nucleic acid sequence having about 95% sequence identity to SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52. The polynucleotide may comprise a nucleic acid sequence having about 96% sequence identity to SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52. The polynucleotide may comprise a nucleic acid sequence having about 97% sequence identity to SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52. The polynucleotide may comprise a nucleic acid sequence having about 98% sequence identity to SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52. The polynucleotide may comprise a nucleic acid sequence having about 99% sequence identity to SEQ ID NO:50, SEQ ID NO:51, or SEQ ID NO:52. The polynucleotide may include SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52. The polynucleotide may consist of SEQ ID NO: 50, SEQ ID NO: 51, or SEQ ID NO: 52.

In some embodiments, the polynucleotide encoding CAR, the polynucleotide encoding IL-15, and optionally the polynucleotide encoding CCL19 are polynucleotides encoding a self-cleaving 2A peptide (2A peptide) or an internal ribosome entry site (IRES). Each is independently transcribed under a promoter containing nucleotides. In some embodiments, the polynucleotide encoding IL-15 and the polynucleotide encoding CCL19 are each independently transcribed under a promoter comprising a polynucleotide encoding a 2A peptide or an IRES. In some embodiments, the polynucleotide encoding IL-15 is transcribed under a promoter comprising a polynucleotide encoding a 2A peptide or an IRES.

In some embodiments, the polynucleotide encoding CAR, the polynucleotide encoding IL-15, and optionally the polynucleotide encoding CCL19 are each independently transcribed under a promoter comprising a polynucleotide encoding a 2A peptide. The 2A peptide family has four members: P2A, E2A, F2A and T2A. P2A is derived from porcine tescovirus-1 2A. E2A is derived from equine rhinitis A virus. F2A is derived from foot-and-mouth disease virus 18. T2A is derived from thosea asigna virus 2A. Exemplary sequences for 2A peptide members include:

P2A-ATNFSLLKQAGDVEENPGP;

E2A-QCTNYALLKLAGDVESNPGP;

F2A - VKQTLNFDLLKLAGDVESNPGP; and

T2A-EGRGSLLTCGDVEENPGP.

In some embodiments, a peptide linker is further added to the end of the 2A peptide, such as the N-terminus. In some embodiments, the peptide linker comprises GSG. In some cases, P2A includes GSGATNSLLKQAGDVEENPGP (SEQ ID NO: 5).

In some embodiments, the polynucleotide encoding CAR, the polynucleotide encoding IL-15, and optionally the polynucleotide encoding CCL19 are each independently transcribed under a promoter comprising a polynucleotide encoding a 2A peptide. In some cases, the polynucleotide encoding IL-15 and the polynucleotide encoding CCL19 are each transcribed under a promoter containing the same 2A peptide. In some cases, the polynucleotide encoding IL-15 and the polynucleotide encoding CCL19 are each transcribed under a promoter comprising two different 2A peptides. In some cases, the polynucleotide encoding CAR, the polynucleotide encoding IL-15, and the polynucleotide encoding CCL19 are each independently transcribed under promoters comprising different 2A peptides.

In some embodiments, the polynucleotide encoding CAR, the polynucleotide encoding IL-15, and optionally the polynucleotide encoding CCL19 are each independently transcribed under a promoter comprising a polynucleotide encoding a P2A peptide. The P2A peptide may include ATNFSLLKQAGDVEENPGP. The P2A peptide may also include SEQ ID NO:5.

In some embodiments, the polynucleotide encoding CAR and the polynucleotide encoding IL-15 are arranged from 5' end to 3' end in the nucleic acid molecule as follows:

A polynucleotide encoding CAR - a polynucleotide encoding IL-15; or

Polynucleotide encoding IL-15 - Polynucleotide encoding CAR.

In some embodiments, the polynucleotide encoding CAR, the polynucleotide encoding IL-15, and the polynucleotide encoding CCL19 are arranged in the nucleic acid molecule from 5' end to 3' end as follows:

a polynucleotide encoding CAR - a polynucleotide encoding IL-15 - a polynucleotide encoding CCL19;

a polynucleotide encoding CAR - a polynucleotide encoding CCL19 - a polynucleotide encoding IL-15;

A polynucleotide encoding IL-15 - a polynucleotide encoding CAR - a polynucleotide encoding CCL19;

A polynucleotide encoding CCL19 - a polynucleotide encoding CAR - a polynucleotide encoding IL-15;

a polynucleotide encoding IL-15 - a polynucleotide encoding CCL19 - a polynucleotide encoding CAR; or

Polynucleotide encoding CCL19 - Polynucleotide encoding IL-15 - Polynucleotide encoding CAR.

In some embodiments, the isolated nucleic acid molecule comprises a polynucleotide encoding mIL-15/Ra and a polynucleotide encoding CCL19. 일부 경우에, 단리된 핵산 분자는 MYRMQLLSCIALSLALVTNSATSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS SGGGSGGGGSGGGGSGGGGSGGGSLQ ITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHLGSGATNFSLLKQAGDVEENPGPMALLLALSLLVLWTSPAPTLSGTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSS (서열번호 14)(또한 본원에서 mIL15/Ra-P2A-CCL19로서 지칭됨)에 대해 약 80%, 85%, 90%, 95%, 96%, Encodes a polypeptide comprising 97%, 98%, 99%, or 100% sequence identity. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 85% sequence identity to SEQ ID NO:14. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:14. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:14. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:14. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:14. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 98% sequence identity to SEQ ID NO:14. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:14. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO:14. In some cases, the isolated nucleic acid molecule encodes a polypeptide consisting of SEQ ID NO:14.

In some embodiments, the isolated nucleic acid molecule comprises a polynucleotide encoding sushi15 and a polynucleotide encoding CCL19. 일부 경우에, 단리된 핵산 분자는 MAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR SGGGSGGGGSGGGGSGGGGSGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSGATNFSLLKQAGDVEENPGPMALLLALSLLVLWTSPAPTLSGTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSS (서열번호 31)(또한 본원에서 IL15Rasp-sushi15-P2A-endosp-CCL19 또는 sushi15-CCL19로서 지칭됨)에 대해 약 80%, 85%, 90%, Encodes a polypeptide comprising 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 85% sequence identity to SEQ ID NO:31. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:31. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:31. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:31. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 97% sequence identity to SEQ ID NO:31. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:31. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:31. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO:31. In some cases, the isolated nucleic acid molecule encodes a polypeptide consisting of SEQ ID NO:31.

In some embodiments, the isolated nucleic acid molecule comprises a polynucleotide encoding a CAR that specifically binds mesothelin and a polynucleotide encoding mIL-15/Ra. 일부 경우에, 단리된 핵산 분자는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSSGILGSGGGGSGGGGSGGGGSQPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSRAAAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYC N KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMYRMQLLSCIALSLALVTNS ATS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHL (서열번호 34)(또한 본원에서 ssVH-P4-CAR-P2A-IL2sp-mIL15/Ra 또는 P4-BB-mIL15/Ra로서 지칭됨)에 대해 encodes a polypeptide comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 85% sequence identity to SEQ ID NO:34. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:34. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:34. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:34. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 97% sequence identity to SEQ ID NO:34. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:34. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:34. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO:34. In some cases, the isolated nucleic acid molecule encodes a polypeptide consisting of SEQ ID NO:34.

In some embodiments, the isolated nucleic acid molecule encoding ssVH-P4-CAR-P2A-IL2sp-mIL15/Ra or P4-BB-mIL15/Ra is ATGGACTGGACATGGCGGATACTCTTCCTCGTCCGCTGCTGCAACCGGAGCCCACAGCCAGGTGCAGCTCCAGCAGTCTGGGCCAGGTTTGGTGACTCCTAGTCAGACATTGAGCTTGACTTGTGCTATCAGCGGAGACTCTGTTT CATCTAATTCTGCAACTTGGAACTGGATTCGGCAGTCCCCCAGCCGGGGGCTCGAGTGGTTGGGTCGGACCTACTATCGGAGCAAATGGTACAATGACTATGCAGTGAGCGTCAAATCAAGAATGAGCATCAATCCTGACACAAGCAAGAACCAGTTTAGCCTTCAGCTTAATAGCGTGACTCCAGAGGACACAGCTGTGTACTATTGCGCGAGAGGCATGATGACATACTATTACGGAATGGACGTGTGGGGCCAGGGAACTACTGT TACAGTGTCAAGCGGAATCCTCGGTAGCGGAGGCGGCGGTTCCGGCGGAGGGGGTAGTGGTGGCGGGGGTAGTCAACCTGTGCTGACCCAGAGCAGCTCTCTTAGTGCTAGCCCAGGTGCAAGTGCAAGTCTTACCTGTACACTGCGCTCCGGTATTAATGTGGGCCCTTACCGAATTTACTGGTACCAGCAGAAACCAGGCTCCCCTCCCCAGTATCTGCTGAACTATAAGTCTGACTCAGACAAACAGCAGGGCTCCGGT GTGCCATCCCGATTTAGTGGCTCAAAGGATGCTAGTGCAAATGCCGGTGTTCTCCTGATCAGCGGACTCAGATCAGAGGACGAAGCAGACTATTACTGTATGATTTGGCATAGCAGCGCTGCTGTCTTCGGAGGAGGGACTCAGCTCACTGTCTTGAGTCGGGCCGCTGCACCTACCACTACCCCTGCCCCTCGACCCCCTACTCCCGCCCCAACTATCGCATCCCAACCACTCTCTCTCAGACCCGAAGCCTGTAGACCCGCAG CCGGTGGCGCTGTGCATACTCGCGGACTTGATTTTGCTTGTGATATTTATATCTGGGCCCCCCTTGCCGGAACTTGTGGAGTTCTCCTGCTGTCTCTCGTAATCACCCTTTATTGTAACAAACGGGGGCGCAAAAAACTTCTTTACATTTTCAAGCAGCCCTTTATGCGGCCCGTGCAGACCACACAGGAAGAAGATGGCTGCAGCTGCAGGTTCCCAGAGGAAGAAGAGGGCGGCTGCGAGCTGCGAGTAAAGTTCAGCC GGAGCGCCGATGCACCTGCATACCAGCAGGGTCAGAACCAGCTCTACAATGAGCTGAACCTGGGCAGAAGAGAGGAATATGATGTACTCGACAAGAGAAGGGAGAGGAATATGATGTACTCGACAAGAGAAGGGGACGCGATCCAGAGATGGGCGGCAAACCACGGCGGAAAAATCCGCAGGAGGGGCTCTATAACGAGCTCCAGAAGGACAAGATGGCAGAAGCCTACTCAGAAATTGGCATGAAAGGAGAGAGAAGGAGGGGAAAGGGCCATGATGGCCTTTACCAAGGGTT GTCTACTGCCACCAAGGATACGTACGATGCACTCCATATGCAGGCTCTTCCTCCCCGAGGTTCAGGCGCAACAAATTTTTCACTTCTTAAACAAGCTGGCGATGTCGAGGAAAACCCAGGTCCCATGTATAGAATGCAGCTTCTGTCATGTATCGCACTGAGTCTGGGCCCTGGTGACCAACAGTGCCACCAGCAACTGGGTGAATGTGATAAGCGACCTTAAGAAAATAGAAGACCTTATTCAGTCCATGCACATAGATGCCACTG TACACCGAGAGCGATGTGCACCCTTCCTGCAAAGTGACAGCTATGAAATGCTTCCTTCTGGAACTGCAAGTAATTTCATTGGAATCTGGCGATGCTTCCATACATGACACCGTGGAAAACCTTATTATTTTGGCTAACAATTCATTGAGCTCAAATGGAAACGTGACAGAATCCGGTTGTAAGGAATGTGAAGAGCTGGAAGAAAAAAATATCAAGGAATTCCTGCAGAGCTTTGTTCACATTGTGCAAATGTTTATTAATACATCC TCAGGGGGCGGTTCCGGAGGCGGGGGAAGTGGCGGAGGAGGAAGCGGCGGAGGAGGAAGCGGAGGAGGATCACTTCAAATCACATGTCCCCCCCCTATGAGTGTTGAACATGCTGACATCTGGGTGAAATCCTATTCCCTTTATTCAAGAGAACGATACATATGTAATTCCGGGTTTAAGAGGAAAGCAGGCACATCATCTCTCACCGAATGTGTCCTGAATAAGGCGACAAACGTAGCTCACTGGACTACGCCCTCCC TCAAATGCATTAGAGACCCAGCACTCGTGCACCAAAGGCCAGCCCCCCCAAGCACCGTCACTACTGCAGGTGTAACCCCGCAACCAGAATCCCTCTCACCAAGCGGAAAAGAGCCAGCCGCATCTTCTCCTAGTTCCAATAATACAGCCGCGACAACAGCCGCAATTGTCCCTGGAAGCCAGTTGATGCCATCAAAGTCCCCAAGTACGGGTACGACCGAAATCTCCTCCCCACGAAAGCAGCCACGGAACACCAAGCCAGACTACCGCCAA GAACTGGGAGCTGACCGCTTCTGCATCACATCAGCCGCCGGGAGTGTATCCACAGGGGCACTCTGATACCACAGTAGCAATCTCAACCTCCACCGTCCTGCTGTGTGGCCTTAGCGCTGTGTCTCTCCTCGCATGTTACCTCAAATCCAGGCAGACCCCCCCCCTTGCTAGTGTCGAAATGGAGGCAATGGAAGCACTTCCCGTGACATGGGGCACTTCTAGCAGAGATGAGGACCTTGAAAACTGCTCCACCACCTC ( About 80%, 85%, 90 for SEQ ID NO: 37) %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 85% sequence identity to SEQ ID NO:37. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 90% sequence identity to SEQ ID NO:37. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 95% sequence identity to SEQ ID NO:37. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 96% sequence identity to SEQ ID NO:37. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 97% sequence identity to SEQ ID NO:37. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 98% sequence identity to SEQ ID NO:37. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 99% sequence identity to SEQ ID NO:37. In some cases, the isolated nucleic acid molecule includes SEQ ID NO:37. In some cases, the isolated nucleic acid molecule consists of SEQ ID NO:37.

In some embodiments, the isolated nucleic acid molecule comprises a polynucleotide encoding a CAR that specifically binds mesothelin and a polynucleotide encoding sushi15. In some cases, the isolated nucleic acid molecule is: SLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSRAAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTELNCV KATNVAHWTTPPSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (SEQ ID NO: 35) (also herein referred to as ssVH-P4-CAR-P2A-sushi15 or P (referred to as 4-BB-sushi15) about 80%, 85%, 90%, Encodes a polypeptide comprising 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 85% sequence identity to SEQ ID NO:35. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:35. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:35. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 96% sequence identity to SEQ ID NO:35. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:35. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:35. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:35. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO:35. In some cases, the isolated nucleic acid molecule encodes a polypeptide consisting of SEQ ID NO:35.

In some embodiments, the isolated nucleic acid molecule encoding ssVH-P4-CAR-P2A-sushi15 or P4-BB-sushi15 is ATGGACTGGACATGGCGGATACTCTTCCTCGTCGCTGCTGCAACCGGAGCCCACAGCCAGGTGCAGCTCCAGCAGTCTGGGCCAGGTTTGTGACTCCTAGTCAGACATTGAGCTTGACTTGTGCTATCAGCGGAGACTCTGTTTCATCTAATTCTGCAACT TGGAACTGGATTCGGCAGTCCCCCAGCCGGGGGCTCGAGTGGTTGGGTCGGACCTACTATCGGAGCAAATGGTACAATGACTATGCAGTGAGCGTCAAATCAAGAATGAGCATCAATCCTGACACAAGCAAGAACCAGTTTAGCCTTCAGCTTAATAGCGTGACTCCAGAGGACACAGCTGTGTACTATTGCGCGAGAGGCATGATGACATACTATTACGGAATGGACGTGTGGGGCCAGGGAACTACTGTTACAGTGTCAAGCGGAA TCCTCGGTAGCGGAGGCGGCGGTTCCGGCGGAGGGGGTAGTGGTGGCGGGGGTAGTCAACCTGTGCTGACCCAGAGCAGCTCTCTTAGTGCTAGCCCAGGTGCAAGTGCAAGTCTTACCTGTACACTGCGCTCCGGTATTAATGTGGGCCCTTACCGAATTTACTGGTACCAGCAGAAACCAGGATCCCCTCCCCAGTATCTGCTGAACTATAAGTCTGACTCAGACAAACAGCAGGGCTCCGGTGTGCCATCCCGATTTAG TGGCTCAAAGGATGCTAGTGCAAATGCCGGTGTTCTCCTGATCAGCGGACTCAGATCAGAGGACGAAGCAGACTATTACTGTATGATTTGGCATAGCAGCGCTGCTGTCTTCGGAGGAGGGACTCAGCTCACTGTCTTGAGTCGGGCCGCTGCACCTACCACTACCCCTGCCCCTCGACCCCCTACTCCCGCCCCAACTATCGCATCCCAACCACTCTCTCTCAGACCCGAAGCCTGTAGACCCGCAGCCGGCATTGGCGCTGTGTG ACTCGCGGACTTGATTTTGCTTGTGATATTTATATCTGGGCCCCCCTTGCCGGAACTTGTGGAGTTCTCCTGCTGTCTCTCGTAATCACCCTTTATTGTAACAAACGGGGGCGCAAAAAACTTCTTTACATTTTTCAAGCAGCCCTTTATGCGGCCCGTGCAGACCACACAGGAAGAAGATGGCTGCAGCTGCAGGTTCCCAGAGGAAGAAGAGGGCGGCTGCGAGCTGCGAGTAAAGTTCAGCCGGAGCGCCGATGCAC CTGCATACCAGCAGGGTCAGAACCAGCTCTACAATGAGCTGAACCTGGGCAGAAGAGAGGAATATGATGTACTCGACAAGAGAAGGGGACGCGATCCAGAGATGGGCGGCAAACCACGGCGGAAAAATCCGCAGGAGGGGCTCTATAACGAGCTCCAGAAGGACAAGATGGCAGAAGCCTACTCAGAAATTGGCATGAAAGGAGAGAGAAGGAGGGGAAAGGGCCATGATGGCCTTTACCAAGGGTTGTCTACTGCCACCAAGG ATACGTACGATGCACTCCATATGCAGGCTCTTCCTCCCCGAGGGTCTGGCGCTACGAATTTCTCTCTCCCTTAAACAGGCCGGAGACGTGGAAGAAAATCCCGGCCCGATGGCGCCCCGCAGGGCCCGCGGGTGTCGAACATTGGGTCTGCTCTCTCTTGCTGCTGTTGCTTCTCAGACCTCCCGCCACACGCGGAATTACGTGCCCTCCCCCCATGTCTGTGGAACATGCCGACATATGGGTCAAGTCTTACAGTCTTT ACTCTAGAGAACGGTATATCTGCAATAGCGGGTTCAAAAGAAAAGCAGGGACTTCCAGCCTGACAGAGTGCGTACTGAATAAGGCCACTAACGTTGCTCACTGGACCACCCCATCATTGAAGTGTATTCGATCAGGAGGCGGAAGCGGTGGTGGGGTCAGGGGGTGGCGGTAGTGGAGGCGGGGGCAGCGGAGGGGGCTCTTTGCAAAACTGGGTTAATGTTATTAGCGACCTTAAGAAAATCGAGGACCTGATACAGTCCA TGCACATCGATGCGACCCTGTACACTGAGAGCGATGTGCATCCCAGTTGCAAAGTGACTGCTATGAAATGCTTTCTGCTCGAGTTGCAGGTGATCTCCCTGGAAAGCGGCGACGCCTCAATACACGACACGGTCGAAAATCTGATCATTCTCGCCAACAACTCTCTCTCAAGTAACGGGAATGTGACAGAAAGTGGATGCAAAGAATGCGAGGAACTTGAGGAGAAAAACATTAAAGAATTCCTCCAGTCCTTCGTCCACATCGTGCAGA About 80%, 85%, 90%, 95%, 96 for TGTTTATCAATACTTCC (SEQ ID NO: 38) %, 97%, 98%, 99%, or 100% sequence identity. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 85% sequence identity to SEQ ID NO:38. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 90% sequence identity to SEQ ID NO:38. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 95% sequence identity to SEQ ID NO:38. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 96% sequence identity to SEQ ID NO:38. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 97% sequence identity to SEQ ID NO:38. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 98% sequence identity to SEQ ID NO:38. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 99% sequence identity to SEQ ID NO:38. In some cases, the isolated nucleic acid molecule includes SEQ ID NO:38. In some cases, the isolated nucleic acid molecule consists of SEQ ID NO:38.

In some embodiments, the isolated nucleic acid molecule comprises a polynucleotide encoding a CAR that specifically binds mesothelin, a polynucleotide encoding mIL-15/Ra, and a polynucleotide encoding CCL19. 일부 경우에, 단리된 핵산 분자는 ATS NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSSGGGSGGGGSGGGGSGGGGSGGGSLQITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPSTVTTAGVTPQPESLSPSGKEPAASSPSSNNTAATTAAIVPGSQLMPSKSPSTGTTEISSHESSHGTPSQTTAKNWELTASASHQPPGVYPQGHSDTTVAISTSTVLLCGLSAVSLLACYLKSRQTPPLASVEMEAMEALPVTWGTSSRDEDLENCSHHLGSGATNFSLLKQAGDVEENPGPMALLLALSLLVLWTSPAPTLSGTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSS (서열번호 36)(또한 본원에서 ssVH-P4-CAR-P2A-IL2sp-mIL15/Ra-P2A-CCL19 또는 P4-BB-mIL15-CCL19로서 지칭됨)에 encodes a polypeptide comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 85% sequence identity to SEQ ID NO:36. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:36. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:36. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:36. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:36. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:36. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:36. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO:36. In some cases, the isolated nucleic acid molecule encodes a polypeptide consisting of SEQ ID NO:36.

In some embodiments, the isolated nucleic acid molecule encoding ssVH-P4-CAR-P2A-IL2sp-mIL15/Ra-P2A-CCL19 or P4-BB-mIL15-CCL19 is ATGGATTGGACCTGGCGAATACTCTTCCTCGTCGCAGCGGCCACTGGTGCCCATTCACAAGTCCAACTGCAGCAGAGCGGACCTGGCCTGGTGACACCCAGTCAGACTCTCAGCCTGACT TGTGCAATCAGCGGCGATAGTGTGTCTAGTAATTCTGCAACATGGAACTGGATCAGACAATCACCAAGTCGGGGACTGGAGTGGCTCGGTAGAACCTATTATAGGTCCAAATGGTATAACGATTATGCAGTGTCCGTGAAGTCCCGAATGTCTATCAACCCTGATACTAGTAAGAATCAATTCAGTCTGCAGCTTAACAGCGTAACCCCCGAAGATACTGCTGTGTATTACTGTGCCCGGGGTATGATGACTACTACTACTACTACTACTACT GGAATGGATGTGTGGGGGCAGGGAACAACCGTTACTGTTTCATCCGGCATTCTCGGGAGCGGAGGCGGTGGAAGCGGTGGGGGAGGGTCCGGGGGAGGAGGATCTCAGCCTGTTCTTACTCAATCTTCTTCCCTCTCCGCCTCACCCGGGGCCTCCGCCTCACTGACCTGCACTCTGCGATCAGGCATCAACGTTGGGCCTTATAGAATCTACTGGTACCAGCAAAAGCCTGGATCACCGCCCCAGTACCTGCTGAACT ATAAATCAGACTCAGACAAGCAGCAGGGCTCCGGCGTGCCGAGTCGATTTAGCGGGAGCAAGGACGCGTCTGCTAATGCCGGCGTGCTTCTCATCAGCGGGCTCCGCAGTGAGGATGAGGCAGATTACTACTGCATGATTTGGCATAGCAGTGCAGCCGTATTTGGCGGAGGAACACAGCTGACTGTCCTCTCTCGCGCCGCCGCTCCGACCACCACCCCTGCACCACGCCCACCTACTCCTGCGCCAACCATTGCCAGCCA GCCTCTCTCTCTCCGACCCGAGGCCTGTAGACCTGCCGCTGGCGGTGCAGTTCATACTCGGGGTCTCGATTTCGCCTGCGACATCTACATCTGGGCACCACTGGCTGGCACTTGTGGCGTTTTGCTCCTGTCCCTGGTGATCACTCTCTACTGTAATAAGAGGGGGAGGAAGAAACTCCTGTATATTTTCAAACAACCCTTTATGCGCCCTTGTCCAAACCACCCAGGAAGAAGATGGATGTAGTTGCAGATTCCCAGAA GAAGAGGAGGGTGGGTGTGAACTTAGGGTGAAGTTTAGTCGCAGTGCCGACGCTCCCGCTTACCAACAGGGTCAGAACCAACTCTACAATGAGCTGAATCTGGGGAGGCGCGAAGAATACGACGTTTCTGGATAAAAGACGCGGCCGCGACCCCGAGATGGGCGGGAAACCGCGGAGAAAGAACCCACAGGAAGGATTGTACAATGAGCTCCAGAAAGATAAGATGGCAGAAGCCTACTCCGAGATCGGCATGAAGGGGG AGCGAAGGCGCGGGAAAGGACACGATGGGCTGTACCAGGGTCTTTCAACCGCGACAAAGGACACCTATGATGCTCTCCATATGCAGGCCCTCCCGCCACGCGGAAGTGGAGCAACTAATTTTAGCCTTCTGAAACAAGCTGGCGATGTTGAGGAAAATCCTGGGCCGATGTACAGGATGCAGCTGCTTTCTTGCATTGCACTGAGTTTGGCACTCGTCACCAACTCTGCCACATCAAATTGGGTTAACGTTATCAGCGATC TGAAGAAAATCGAGGATTTGATCCAGAGTATGCATATTGACGCAACCTTGTATACAGAATCTGATGTGCACCCAAGCTGTAAAGTCACAGCTATGAAATGCTTTTTGCTGGAACTCCAAGTGATCTCCCTCGAATCCGGCGATGCATCCATCCACGATACTGTCGAAAACCTTATAATTTTGGCCAATAACAGCCTCAGCAGCAATGGCAACGTGCAGAGTCTGGGTGTAAGGAGTGTGAAGAACTGGAGGAGAAAAATATTAAAATTAAAGAATT CCTGCAGTCCTTTGTACACATTGTGCAAATGTTCATTAACACTTCAAGTGGCGGCGGGAGCGGCGGGGGTGGTTCAGGTGGTGGCGGCAGCGGTGGTGGGGGGTCTGGCGGGGGTAGTCTCCAAATTACTTGTCCTCCCCCAATGAGCGTTGAACACGCCGACATTTGGGTCAAGTCTTATTCACTGTACAGCCGAGAAAGATATATCTGTAACTCTGGATTTAAGCGCAAGGCCGGAACGTCTAGTCTGACTGAGTGCG TGCTGAATAAGGCCACTAATGTTGCCCACTGGACTACCCCCAGCCTGAAGTGTATTCGCGATCCTGCCTTGGTGCACCAACGACCCGCGCCACCCAGCACAGTCACTACTGCCGGTGTGACTCCACAGCCCGAGTCTTTGTCCCCGAGCGGAAAGGAGCCCGCCGCATCTTCACCTTCTTCAAATAACACGGCCGCCACAACCGCTGCAATCGTCCCAGGTAGTCAACTGATGCCCTCTAAAAGCCCCTCTACGGGGACAACTGAG ATAAGCAGCCACGAGTCTAGTCACGGCACACCAAGCCAGACTACCGCCAAAAACTGGGAGCTGACCGCCTCTGCCTCACACCAACCACCAGGCGTGTATCCCCAGGGGCACAGCGACACCACTGTGGCAATCAGCACCAGCACGGTACTGTTGTGCGGACTCTCTGCCGTCAGTCTGCTGGCCTGCTACCTGAAATCCAGACAGACTCCCCCCCTGGCCAGCGTGGAAATGGAAGCTATGGAGGGCTCTGCCCGTGACCTGGG GGACTAGCTCCAGAGATGAAGACTTGGAGAACTGCAGTCACCATCTCGGGTCCGGAGCCACGAATTTCTCTCTCCTCAAACAAGCTGGGGATGTTGAGGAGAACCCTGGGCCAATGGCCCTCTTGCTCGCACTGTCCCTCCTGGTCCTGTGGACATCACCCGCCCCCACCCTGTCCGGCACGAATGACGCAGAAGACTGCTGCCTGTCTGTCACGCAGAAACCCATCCCCGGCTATATAGTGCGGAACTTCCATTACCTGCT About 80% for GATCAAGGACGGATGTAGGGTGCCAGCCGTCGTCTTCACCACCCTGCGAGGGCGCCAGCTGTGCGCTCCTCCTGACCAGCCCTGGGTGGAGCGGATCATTCAACGCTTGCAGCGCACCTCAGCAAAAATGAAAAGAAGAAGTAGT (SEQ ID NO: 39); Includes sequences comprising 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 85% sequence identity to SEQ ID NO:39. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 90% sequence identity to SEQ ID NO:39. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 95% sequence identity to SEQ ID NO:39. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 96% sequence identity to SEQ ID NO:39. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 97% sequence identity to SEQ ID NO:39. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 98% sequence identity to SEQ ID NO:39. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 99% sequence identity to SEQ ID NO:39. In some cases, the isolated nucleic acid molecule includes SEQ ID NO:39. In some cases, the isolated nucleic acid molecule consists of SEQ ID NO:39.

In some embodiments, the isolated nucleic acid molecule comprises a polynucleotide encoding a CAR that specifically binds mesothelin, a polynucleotide encoding sushi 15, and a polynucleotide encoding CCL19. In some cases, the isolated nucleic acid molecule is: SLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLSRAAPTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTELNCV KATNVAHWTTPPSLKCIRSGGGSGGGGSGGGGSGGGGSGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSGATNFSLLKQAGDVEENPGPMALLLALSLLVLWTSPAPTLSGTNDAEDCCLSVTQKPIPGY About 80% for IVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRSS (SEQ ID NO: 15) (also referred to herein as ssVH-P4-CAR-P2A-sushi15-P2A-CCL19 or P4-BB-sushi15-CCL19) Encodes a polypeptide comprising 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 85% sequence identity to SEQ ID NO:15. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:15. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:15. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 96% sequence identity to SEQ ID NO:15. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:15. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 98% sequence identity to SEQ ID NO:15. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:15. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO:15. In some cases, the isolated nucleic acid molecule encodes a polypeptide consisting of SEQ ID NO:15.

In some embodiments, the isolated nucleic acid molecule encoding ssVH-P4-CAR-P2A-sushi15-P2A-CCL19 or P4-BB-sushi15-CCL19 is ATGGATTGGACCTGGCGAATACTCTTCCTCGTCGCAGCGGCCACTGGTGCCCATTCACAAGTCCAACTGCAGCAGAGCGGACCTGGCCTGGTGACACCCAGTCAGACTCTCAGCCTGACTTGTGCAATCAGCG GCGATAGTGTGTCTAGTAATTCTGCAACATGGAACTGGATCAGACAATCACCAAGTCGGGGACTGGAGTGGCTCGGTAGAACCTATTATAGGTCCAAATGGTATAACGATTATGCAGTGTCCGTGAAGTCCCGAATGTCTATCAACCCTGATACTAGTAAGAATCAATTCAGTCTGCAGCTTAACAGCGTAACCCCCGAAGATACTGCTGTGTATTACTGTGCCCGGGGTATGATGACTTACTACTACGGAATGGATGT GTGGGGGCAGGGAACAACCGTTACTGTTTCATCCGGCATTCTCGGGAGCGGAGGCGGTGGAAGCGGTGGGGGAGGGTCCGGGGGAGGAGGATCTCAGCCTGTTCTTACTCAATCTTCTTCCCTCTCCGCCTCACCCGGGGCCTCCGCCTCACTGACCTGCACTCTGCGATCAGGCATCAACGTTGGGCCTTATAGAATCTACTGGTACCAGCAAAAGCCTGGATCACCGCCCCAGTACCTGCTGAACTATAAATCAGACTCA GACAAGCAGCAGGGCTCCGGCGTGCCGAGTCGATTTAGCGGGAGCAAGGACGCGTCTGCTAATGCCGGCGTGCTTCTCATCAGCGGGCTCCGCAGTGAGGATGAGGCAGATTACTACTGCATGATTTGGCATAGCAGTGCAGCCGTATTTGGCGGAGGAACACAGCTGACTGTCCTCTCTCGCGCCGCCGCTCCGACCACCACCCCTGCACCACGCCCACCTACTCCTGCGCCAACCATTGCCAGCCAGCCTCTCTCTCT CCGACCCGAGGCCTGTAGACCTGCCGCTGGCGGTGCAGTTCATACTCGGGGTCTCGATTTCGCCTGCGACATCTACATCTGGGCACCACTGGCTGGCACTTGTGGCGTTTTGCTCCTGTCCCTGGTGATCACTCTCTACTGTAATAAGAGGGGGAGGAAGAAACTCCTGTATATTTTCAAACAACCCTTTATGCGCCCTGTCCAAACCACCCAGGAAGAAGATGGATGTAGTTGCAGATTCCCAGAAGAAGAGGAGGGT GGGTGTGAACTTAGGGTGAAGTTTAGTCGCAGTGCCGACGCTCCCGCTTACCAACAGGGTCAGAACCAACTCTACAATGAGCTGAATCTGGGGAGGCGCGAAGAATACGACGTTTCTGGATAAAAGACGCGGCCGCGACCCCGAGATGGGCGGGAAACCGCGGAGAAAGAACCCACAGGAAGGATTGTACAATGAGCTCCAGAAAGATAAGATGGCAGAAGCCTACTCCGAGATCGGCATGAAGGGGGAGCGAAGGCGCG GGAAAGGACACGATGGGGCTGTACCAGGGTCTTTCAACCGCGACAAAGGACACCTATGATGCTCTCCATATGCAGGCCCTCCCGCCCACGCGGAAGTGGAGCAACTAATTTTAGCCTTCTGAAACAAGCTGGCGATGTTGAGGAAAATCCTGGGCCGATGGCACCTAGACGGGCACGCGGGTGTAGAACGCTGGGCCTCCCCGCACTGTTGTTGCTCTTGCTTCTGAGACCTCCCGCTACAAGGGGGATAACTTGCCCTCCA CCTATGAGCGTCGAGCATGCTGACATTTGGGTGAAGTCCTATTCACTCTATTCCCGGAGCGGTACATCTGTAACTCTGGATTCAAGAGGAAAGCCGGCACCAGCAGTCTGACCGAGTGCGTGCTGAATAAGGCCACCAATGTGGCCCACTGGACAACCCCTAGCCTTAAATGTATACGGTCAGGGGGCGGATCTGGAGGCGGCGGCTCCGGTGGAGGCGGGAGTGGGGGCGGGGGCTCTGGAGGTGGTAGCCTGCA GAATTGGGTTAACGTGATTAGCGACCTCAAAAAAATCGAAGATCTTATCCAGAGCATGCATATAGACGCAACCCTGTACACAGAAAGCGATGTTCACCCGTCCTGCAAGGTAACGGCTATGAAGTGTTTTCTTTTGGAGTTGCAAGTCATATCACTGGAAAGTGGGGATGCCTCAATTCACGATACCGTGGAGAACCTCATCATCCTCGCAAATAACAGCCTGAGCTCCAATGGCAATGTCACAGAGTCAGGTTGCAAAGAGTGTG AAGAGCTGGAAGAGAAAAACATCAAAGAGTTCCTCCAGTCATTTGTGCACATTGTCCAGATGTTCATTAACACTAGTGGTAGTGGTGCCACAAATTTTAGTCTGTTGAAACAGGCCGGGGACGTCGAAGAAAACCCGGGGCCTATGGCCCTCTTGCTCGCACTGTCCCTCCTGGTCCTGTGGACATCACCCGCCCCCACCCTGTCCGGCACGAATGACGCAGAAGACTGCTGCCTGTCTGTCACGCAGAAACCCATCCCCGGCT ATATAGTGCGGAACTTCCATTACCTGCTGATCAAGGACGGATGTAGGGTGCCAGCCGTCGTCTTCACCACCCTGCGAGGGCGCCAGCTGTGCGCTCCTCCTGACCAGCCCTGGGTGGAGCGGATCATTCAACGCTTGCAGCGCACCTCAGCAAAAATGAAAAGAAGAAGTAGT for about 80%, 85%, 90% (SEQ ID NO: 40) %, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 85% sequence identity to SEQ ID NO:40. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 90% sequence identity to SEQ ID NO:40. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 95% sequence identity to SEQ ID NO:40. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 96% sequence identity to SEQ ID NO:40. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 97% sequence identity to SEQ ID NO:40. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 98% sequence identity to SEQ ID NO:40. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 99% sequence identity to SEQ ID NO:40. In some cases, the isolated nucleic acid molecule includes SEQ ID NO:40. In some cases, the isolated nucleic acid molecule consists of SEQ ID NO:40.

In some embodiments, the isolated nucleic acid molecule comprises a polynucleotide encoding a CAR that specifically binds mesothelin and a polynucleotide encoding mIL15/Ra-LSP. 일부 경우에, 단리된 핵산 분자는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS RAAA PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD IYIWAPLAGTCGVLLLSLVITLYCN (서열번호 53)(또한 본원에서 ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15프로펩티드-mIL15/Ra 또는 ssVHsp- a polypeptide comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to (referred to as P4-BB-mIL15/Ra-LSP) Encode. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 85% sequence identity to SEQ ID NO:53. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:53. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:53. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 96% sequence identity to SEQ ID NO:53. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:53. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 98% sequence identity to SEQ ID NO:53. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:53. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO:53. In some cases, the isolated nucleic acid molecule encodes a polypeptide consisting of SEQ ID NO:53.

In some embodiments, the isolated nucleic acid molecule encoding ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15propeptide-mIL15/Ra or ssVHsp-P4-BB-mIL15/Ra-LSP is ATGGACTGGACATGGCGGATACTCTTCCTCGTCGCTGCTGCAACCGGAGCCCACAGCCAGGTGCAGCTCCAGCAGTCTGGGCCA GGTTTGGTGACTCCTAGTCAGACATTGAG CTTGACTTGTGCTATCAGCGGAGACTCTGTTTCATCTAATTCTGCAACTTGGAACTGGATTCGGCAGTCCCCCAGCCGGGGGCTCGAGTGGTTGGGTCGGACCTACTATCGGAGCAAATGGTACAATGACTATGCAGTGAGCGTCAAATCAAGAATGAGCATCAATCCTGACACAAGCAAGAACCAGTTTAGCCTTCAGCTTAATAGCGTGACTCCAGAGGACACAGCTGTGTACT ATTGCGCGAGAGGCATGATGACATACTATTACGGAATGGACGTGTGGGGCCAGGGAACTACTGTTACAGTGTCAAGCGGAATCCTCGGTAGCGGAGGCGGCGGTTCCGGCGGAGGGGGTAGTGGTGGCGGGGGTAGTCAACCTGTGCTGACCCAGAGCAGCTCTCTTAGTGCTAGCCCAGGTGCAAGTGCAAGTCTTACCTGTACACTGCGCTCCGGTATTAATGTGGGCCCTTACCGAATTTACTGGTACCAGCAGAAAGGCCA CTCCCCTCCCCAGTATCTGCTGAACTATAAGTCTGACTCAGACAAACAGCAGGGCTCCGGTGTGCCATCCCGATTTAGTGGCTCAAAGGATGCTAGTGCAAATGCCGGTGTTCTCCTGATCAGCGGACTCAGATCAGAGGACGAAGCAGACTATTACTGTATGATTTGGCATAGCAGCGCTGCTGTCTTCGGAGGAGGGACTCAGCTCACTGTCTTGAGTCGGGCCGCTGCACCTACCACTACCCCTGCCCCTCGACCCCCT ACTCCCGCCCCAACTATCGCATCCCAACCACTCTCTCTCAGACCCGAAGCCTGTAGACCCGCAGCCGGTGGCGCTGTGCATACTCGCGGACTTGATTTTGCTTGTGATATTTATATCTGGGCCCCCCTTGCCGGAACTTGTGGAGTTCTCCTGCTGTCTCTCGTAATCACCCTTTATTGTAACAAACGGGGGCGCAAAAAACTTCTTTACATTTTCAAGCAGCCCTTTATGCGGCCCGTGCAGACCACACAGGAAGAAGATGGCT GCAGCTGCAGGTTCCCAGAGGAAGAAGAGGGCGGCTGCGAGCTGCGAGTAAAGTTCAGCCGGAGCGCCGATGCACCTGCATACCAGCAGGGTCAGAACCAGCTCTACAATGAGCTGAACCTGGGCAGAAGAGAGGAATATGATGTACTCGACAAGAGAAGGGGAAGAGAGGAATATGATGTACTCAGA AATTGGCATGAAAGGAGAGAGAAGGAGGGGAAAGGGCCATGATGGCCTTTACCAAGGGTTGTCTACTGCCACCAAGGATACGTACGATGCACTCCATATGCAGGCTCTTCCTCCCCGAGGCTCAGGAGCCACCAACTTCTCCCTGCTGAAGCAGGCCGGCGACGTGGAGGAGAACCCAGGTCCTATGAGAATCTCAAAACCCCATCTTAGAAGCATCTCTATACAGTGTTATCTGTGTCTCTTGCTGAACTCCCACTTTTTGA CAGAAGCTGGGATACATGTCTTTATCCTGGGATGTTTTTCCGCCGGGCTCCCTAAAACCGAGGCCAACTGGGTAAACGTAATCTCAGACCTTAAAAAGATTGAGGACCTTGATTCAGTCAATGCATATCGATGCAACTTTGTACACGGAGAGCGATGTTCACCCAAGTTGTAAAGTGACCGCGATGAAATGTTTTCTCCTCGAATTGCAGGTGATCCCTCGAGTCAGGCGACGCGTCTATCCACGATACTGTGGAAAACCTTA TCATTTTGGCGAACAATAGCCTCTCATCTAATGGTAACGTGACCGAGTCCGGCTGCAAGGAATGTGAGGAACTGGAGGAGAAAAATATCAAGGAATTCCTGCAGTCATTTGTACACATCGTGCAAATGTTTATCAACACTTCTTCAGGAGGCGGGTCAGGAGGGGGAGGCTCAGGCGGCGGAGGTAGTGGAGGAGGAGGAAGTGGAGGCGGCAGTCTCCAGATCACCTGTCCACCACCAATGAGTGTGGAACACGCGGATT GGGTCAAGTCATATTCTCTTTACTCCAGAGAGCGATACATATGCAACAGTGGTTTCAAGCGGAAAGCGGGTACTTCTTCACTTACCGAGTGCGTGCTCAATAAAGCAACCAACGTCGCGCACTGGACAACACCTAGCCTGAAATGCATAAGAGATCCTGCCCTGTTCACCAGCGGCCAGCGCCACCGTCCACAGTGACAACAGCTGGTGTGACACCCCAGCCGGAGAGCCTTAGCCCTAGCGGGCAAAGAGCCGGCCGCAAGCTC ACCAAGCTCAAATAACACAGCCGCGACAACTGCTGCTATCGTGCCCGGTTCACAATTGATGCCGAGCAAATCACCAAGCACCGGAACTACCGAAATCTCAAGTCATGAAAGTAGTCACGGTACTCCTAGCCAGACGACGGCAAAGAATTGGGAGCTGACTGCCTCTGCGAGCCACCAGCCGCCGGGTGTTTACCCTCAGGGGCATTCAGATACTACTGTGGCTATCTCTACTTCCACCGTCCTCTTGTGCGGCTTGTCTGCTG TGTCTCTTCTGGCTTGCTATTTGAAAAGTAGACAGACACCACCCCTTGCAAGTGTCGAGATGGAAGCGATGGAGGCATTGCCTGTGACCTGGGGAACCAGTAGTAGGGACGAGGACCTGGA and a sequence comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to AAATTGTAGTAGCACCACCTGTGA (SEQ ID NO: 58). In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 85% sequence identity to SEQ ID NO:58. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 90% sequence identity to SEQ ID NO:58. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 95% sequence identity to SEQ ID NO:58. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 96% sequence identity to SEQ ID NO:58. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 97% sequence identity to SEQ ID NO:58. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 98% sequence identity to SEQ ID NO:58. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 99% sequence identity to SEQ ID NO:58. In some cases, the isolated nucleic acid molecule includes SEQ ID NO:58. In some cases, the isolated nucleic acid molecule consists of SEQ ID NO:58.

In some embodiments, the isolated nucleic acid molecule comprises a polynucleotide encoding a CAR that specifically binds mesothelin and a polynucleotide encoding sIL15-LSP. 일부 경우에, 단리된 핵산 분자는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS RAAA PTTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSLVITLYCNKRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTS (서열번호 54)(또한 본원에서 ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15프로펩티드-IL15 또는 ssVHsp-P4-BB -sIL15-LSP). In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 85% sequence identity to SEQ ID NO:54. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:54. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:54. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:54. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:54. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:54. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:54. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO:54. In some cases, the isolated nucleic acid molecule encodes a polypeptide consisting of SEQ ID NO:54.

In some embodiments, the isolated nucleic acid molecule encoding ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15propeptide-IL15 or ssVHsp-P4-BB-sIL15-LSP is ATGGACTGGACATGGCGGATACTCTTCCTCGTCGCTGCTGCTGCAACCGGAGCCCACAGCCAGGTGCAGCTCCAGCAGTCTGGGCCAGGTTTTGGTG ACTCCTAGTCAGACATTGAGCTTGACTTGTGCTATCAGCGGAGACTCTGTTTCATCTAATTCTGCAACTTGGAACTGGATTCGGCAGTCCCCCAGCCGGGGGCTCGAGTGGTTGGGTCGGACCTACTATCGGAGCAAATGGTACAATGACTATGCAGTGAGCGTCAAATCAAGAATGAGCATCAATCCTGACACAAGCAAGAACCAGTTTAGCCTTCAGCTTAATAGCGTGACTCCAGAGGACACAGCTGTGTACTATTGCGCGAGA GGCATGATGACATACTATTACGGAATGGACGTGTGGGGCCAGGGAACTACTGTTACAGTGTCAAGCGGAATCCTCGGTAGCGGAGGCGGCGGTTCCGGCGGAGGGGGTAGTGGTGGCGGGGGTAGTCAACCTGTGCTGACCCAGAGCAGCTCTCTTAGTGCTAGCCCAGGTGCAAGTGCAAGTCTTACCTGTACACTGCGCTCCGGTATTAATGTGGGCCCTTACCGAATTTACTGGTACCAGCAGAAACCAGGCTCCCCTCCC CAGTATCTGCTGAACTATAAGTCTGACTCAGACAAACAGCAGGGCTCCGGTGTGCCATCCCGATTTAGTGGCTCAAAGGATGCTAGTGCAAATGCCGGTGTTCTCCTGATCAGCGGACTCAGATCAGAGGACGAAGCAGACTATTACTGTATGATTTGGCATAGCAGCGCTGCTGTCTTCGGAGGAGGGACTCAGCTCACTGTCTTGAGTCGGGCCGCTGCACCTACCACTACCCCTGCCCCTCGACCCCCTACTCCCGCCCCA ACTATCGCATCCCAACCACTCTCTCTCAGACCCGAAGCCTGTAGACCCGCAGCCGGTGGCGCTGTGCATACTCGCGGACTTGATTTTGCTTGTGATATTTATATCTGGGCCCCCCTTGCCGGAACTTGTGGAGTTCTCCTGCTGTCTCTCGTAATCACCCTTTATTGTAACAAACGGGGGCGCAAAAAACTTCTTTACATTTTCAAGCAGCCCTTTATGCGGCCCGTGCAGACCACACAGGAAGAAGATGGCTGCAGCTGCAG GTTCCCAGAGGAAGAAGAGGGCGGCTGCGAGCTGCGAGTAAAGTTCAGCCGGAGCGCCGATGCACCTGCATACCAGCAGGGTCAGAACCAGCTCTACAATGAGCTGAACCTGGGCAGAAGAGAGGAATATGATGTACTCGACAAGAGAAGGGGACGCGATCCAGAGATGGGCGGCAAACCACGGCGGAAAAATCCGCAGGAGGGGCTCTATAACGAGCTCCAGAAGGACAAGATGGCAGAAGCCTACTCAGAAATTGGCATG AAAGGAGAGAGAAGGAGGGGAAAGGGCCATGATGGCCTTTACCAAGGGTTGTCTACTGCCACCAAGGATACGTACGATGCACTCCATATGCAGGCTCTTCCTCCCCGAGGTTCAGGCGCAACAAATTTTTCACTTCTTAAACAAGCTGGCGATGTCGAGGAAAACCCAGGTCCCATGCGGATCTCTAAACCCCACTTGCGGAGCATTTCTATCCAGTGTTATCTTTGCCTCCTGCTTAACTCCCACTTTCTCACAGAAGCAGGGATA CACGTGTTCATCCTGGGCTGTTTTTCTGCCGGTCTCCCCAAAACAGAAGCCAACTGGGTGAATGTGATCAGTGATCTTAAGAAAATAGAAGACCTCATCCAGTCAATGCACATCGATGCCACCTTGTACACTGAGAGCGACGTGCACCCTTCCTGCAAGGTGACAGCTATGAAGTGCTTCCTGCTTGAGCTCCAGGTCATATCCCTTGAGTCCTGGAGATGCAAGTATCCACGATACGTGGAAAACCTTATTATACTGGCCAATAATTC TCTTTCTTCCAATGGCAAATGTTACCGAATCAGGGTGTAAAGAGTGCGAAGAGCTGGAGGAGAAAAATATCAAAGAGTTTTTGCAGTCATTTGTGCACATCGTCCAGATG TTTATTAATACAAGTTGA (SEQ ID NO: 59 ), comprising a sequence comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 85% sequence identity to SEQ ID NO:59. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 90% sequence identity to SEQ ID NO:59. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 95% sequence identity to SEQ ID NO:59. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 96% sequence identity to SEQ ID NO:59. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 97% sequence identity to SEQ ID NO:59. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 98% sequence identity to SEQ ID NO:59. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 99% sequence identity to SEQ ID NO:59. In some cases, the isolated nucleic acid molecule includes SEQ ID NO:59. In some cases, the isolated nucleic acid molecule consists of SEQ ID NO:59.

In some embodiments, the isolated nucleic acid molecule comprises a polynucleotide encoding a CAR that specifically binds mesothelin, a polynucleotide encoding mIL15/Ra-LSP, and a polynucleotide encoding CCL19. In some cases, the isolated nucleic acid molecule is MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASA SLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS RAAA (SEQ ID NO: 55) (also herein referred to as ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15 propeptide-IL15-P2A-endos pCCL19 or ssVHsp- Comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to (referred to as P4-BB-mIL15/Ra-LSP-CCL19) Encodes a polypeptide. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 85% sequence identity to SEQ ID NO:55. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:55. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:55. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 96% sequence identity to SEQ ID NO:55. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:55. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 98% sequence identity to SEQ ID NO:55. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:55. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO:55. In some cases, the isolated nucleic acid molecule encodes a polypeptide consisting of SEQ ID NO:55.

In some embodiments, an isolated nucleic acid encoding ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15propeptide-IL15-P2A-endospCCL19 or ssVHsp-P4-BB-mIL15/Ra-LSP-CCL19 The molecule is ATGGATTGGACCTGGCGAATACTCTTCCTCGTCGCAGCGGCCACTGGTGCCCATTCACAAGTCCAACTGCAGCAGAGCGGACCTGGCCTGGTGACACCCAGTCAGACTCTCAGCCTGACTTGTGCAATCAGCGGCGATAGTGTGTCTAGTAATTCTGCAACATGGAACTGGATCAGACAATCACCAAGTCGGGGACTGGAGTGGCTCGGTAGAACCTATTATAGGTCCAAATGGTATAACGATTATGCAGTGTCC GTGAAGTCCCGAATGTCTATCAACCCTGATACTAGTAAGAATCAATTCAGTCTGCAGCTTAACAGCGTAACCCCCGAAGATACTGCTGTGTATTACTGTGCCCGGGGTATGATGACTTACTACTACGGAATGGATGTGTGGGGGCAGGGAACAACCGTTACTGTTTCATCCGGCATTCTCGGGAGCGGAGGCGGTGGAAGCGGTGGGGGAGGGTCCGGGGGAGGAGGATCTCAGCCTGTTCTTACTCAATCTTCTTC CCTCTCCGCCTCACCCGGGGCCTCCGCCTCACTGACCTGCACTCTGCGATCAGGCATCAACGTTGGGCCTTATAGAATCTACTGGTACCAGCAAAAGCCTGGATCACCGCCCCAGTACCTGCTGAACTATAAATCAGACTCAGACAAGCAGCAGGGCTCCGGCGTGCCGAGTCGATTTAGCGGGAGCAAGGACGCGTCTGCTAATGCCGGCGTGCTTCTCATCAGCGGGCTCCGCAGTGAGGATGAGGCAGATTACTACTG CATGATTTGGCATAGCAGTGCAGCCGTATTTGGCGGAGGAACACAGCTGACTGTCCTCTCTCGCGCCGCCGCTCCGACCACCACCCCTGCACCACGCCCACCTACTCCTGCGCCAACCATTGCCAGCCAGCCTCTCTCTCTCCGACCCGAGGCCTGTAGACCTGCCGCTGGCGGTGCAGTTCATACTCGGGGTCTCGATTTCGCCTGCGACATCTACATCTGGGCACCACTGGCTGGCACTTGTGGCGTTTTGCTCCTGTC CCTGGTGATCACTCTCTACTGTAATAAGAGGGGGAGGAAGAAACTCCTGTATAATTTTCAAACAACCCTTTATGCGCCCTGTCCAAACCACCCAGGAAGAAGATGGATGTAGTTGCAGATTCCCAGAAGAAGAGGAGGGTGGGTGTGAACTTAGGGTGAAGTTTAGTCGCAGTGCCGACGCTCCCGCTTACCAACAGGGTCAGAACCAACTCTACAATGAGCTGAATCTGGGGAGGCGCGAAGAATACGACGTTTCTGG ATAAAAGACGCGGCCGCGACCCCGAGATGGGCGGGAAACCGCGGAGAAAGAACCCACAGGAAGGATTGTACAATGAGCTCCAGAAAGATAAGATGGCAGAAGCCTACTCCGAGATCGGCATGAAGGGGGAGCGAAGGCGCGGGAAAGGACACGATGGGCTGTACCAGGGTCTTTCAACCGCGACAAAGGACACCTATGATGCTCTCCATATGCAGGCCCTCCCGCCACGCGGAAGTGGAGCAACTAATTTTAGCCTTCTG AAACAAGCTGGCGATGTTGAGGAAAATCCTGGGCCGATGCGCATTAGCAAGCCACATCTGAGGAGTATCAGCATCCAGTGCTACCTTTGCCTGCTGCTCAACTCTCACTTTCTGACAGAAGCTGGCATCCACGTCTTCATCCTGGGGTGCTTCAGCGCCGGCTTGCCGAAGACCGAAGCCAACTGGGTGAATGTGATCTCCGACCTCAAGAAGATCGAGGACCTGATCCAGAGTATGCATATTGATGCTACACTTTACACCGAGTC CGATGTTCACCCTAGTTGTAAGGTGACTGCCATGAAATGTTTCTTGCTGGAGCTTCAGGTAATAAGCCTTGAGTTCTGGGGATGCAAGCATTCATGACACGGTTGAGAATCTCATCATCCTGGCAAATAATTCACTGTCTTCAAATGGTAACGTTACAGAGAGCGGCTGTAAGGAGTGCGAAGAGCTTGAAGAGAAAAACATCAAGGAATTCCTCCAGAGTTTCGTGCACATCGGTGCAAATGTTCATCAACACGAGCTCTGGAG GCGGATCAGGAGGCGGAGGATCAGGGGGGGGAGGGTCAGGCGGAGGGGGATCTGGTGGAGGCAGCCTTCAAATCACATGCCCGCCACCTATGTCCGTTGAGCACGCCGACATATGGGTGAAGTCATATTCACTGTATAGTCGGGAGAGGTACATTTGTAATTCAGGTTTCCAAGCGAAAAGCTGGGACATCAAGCCTGACAGAATGCGTACTTAACAAGGCCACAAATGTCGCCCATTGGACCACTCCGAGTCTGAAGTGTATAC GAGATCCCGCACTGGTGCACCAGCGACCTGCTCCCCCTAGTACAGTAACAACCGCGGGCGTTACGCCCCAGCCTGAATCCCTGAGCCCATCTGGCAAGGAGCCTGCAGCTAGCTCTCCGAGCAGCAATAATACTGCAGCGACCACTGCAGCCATCGTCCCCGGCTCCCAGCTCATGCCTAGTAAAAGTCCGTCTACAGGAACGACCGAAATCTCCAGCCACGAGTCTAGTCACGGGACCCCGAGTCAGACCACTGCCAA GAACTGGGAGCTTACGGCCAGTGCCTCCCATCAACCCCCGGGCGTCTACCCGCAAGGCCATAGCGACACCACAGTCGCCATTAGCACATCTACTGTCCTCTTGTGCGGGCTCTCCGCTGTGTCCCTCCTGGCCTGTTATCTCAAGAGCAGACAGACCCCCCCATTGGCAAGCGTTGAGATGGAGGCAATGGAGGCTCTGCCCGTTACTTGGGGGACTTCTTCACGCGACGAGGATCTGGAGAACTGCTCCCACCACCTGGGGT GGTGCCACAAATTTCAGCCTGCTCAAGCAGGCCGGGGATGTTGAAGAGAGAACCCAGGGCCGATGGCCCTCTTGCTCGCACTGTCCCTCCTGGTCCTGTGGACATCACCCGCCCCCACCCTGTCCGGCACGAATGACGCAGAAGACTGCTGCCTGTCTGTCACGCAGAAACCCATCCCCGGCTATATAGTGCGGAACTTCCATTACCTGCTGATCAAGGACGGATGTAGGGTGCCAGCCGTCGTCTTCACCACCCTGCGAGG GCGCCAGCTGTGGGTGGAGCGGATCATTCAACGCTTGCAGCGCACCTCAGCAAAAATGAAAAGAAGAAGTAGTTGA (SEQ ID NO: 60). In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 85% sequence identity to SEQ ID NO:60. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 90% sequence identity to SEQ ID NO:60. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 95% sequence identity to SEQ ID NO:60. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 96% sequence identity to SEQ ID NO:60. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 97% sequence identity to SEQ ID NO:60. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 98% sequence identity to SEQ ID NO:60. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 99% sequence identity to SEQ ID NO:60. In some cases, the isolated nucleic acid molecule includes SEQ ID NO:60. In some cases, the isolated nucleic acid molecule consists of SEQ ID NO:60.

In some embodiments, the isolated nucleic acid molecule comprises a polynucleotide encoding a CAR that specifically binds mesothelin, a polynucleotide encoding sIL15-LSP, and a polynucleotide encoding CCL19. 일부 경우에, 단리된 핵산 분자는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS RAAA P TTTPAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD IYIWAPLAGTCGVLLLSLVITLYCN KRGRKKLLYIFKQPFMRPVQTTQEEDGCSCRFPEEEEGGCELRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMRISKPHLRSISIQCYLCLLLNSHFLTEAGIHVFILGCFSAGLPKTEANWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSGATNFSLLKQAGDVEENPGPMALLLALSLLVLWTSPAPTLSGTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSS (서열번호 56)(또한 본원에서 ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15프로펩티드-IL15-P2A- approximately 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to endospCCL19 (referred to as ssVHsp-P4-BB-sIL15-LSP-CCL19) Encodes a polypeptide containing In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 85% sequence identity to SEQ ID NO:56. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:56. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:56. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:56. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:56. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 98% sequence identity to SEQ ID NO:56. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:56. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO:56. In some cases, the isolated nucleic acid molecule encodes a polypeptide consisting of SEQ ID NO:56.

In some embodiments, the isolated nucleic acid molecule encoding ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15propeptide-IL15-P2A-endospCCL19 or ssVHsp-P4-BB-sIL15-LSP-CCL19 ATGGATTGGACCTGGGCGAATACTCTTCCTCGTCGCAGCGGCCACTGGTGCCCATTCACAAGTCCAACTGCAGCAGAGCGGACCTGGCCTGGTGACACCCAGTCAGACTCTCAGCCTGACTTGTGCAATCAGCGGCGATAGTGTGTCTAGTAATTCTGCAACATGGAACTGGATCAGACAATCACCAAGTCGGGGACTGGAGTGGCTCGGTAGAACCTATTATAGGTCCAAATGGTATAACGATTATGCAGTGTCCGTGA AGTCCCGAATGTCTATCAACCCTGATACTAGTAAGAATCAATTCAGTCTGCAGCTTAACAGCGTAACCCCCGAAGATACTGCTGTGTATTACTGTGCCCGGGGTATGATGACTTACTACTACGGAATGGATGTGTGGGGGCAGGGAACAACCGTTACTGTTTCATCCGGCATTCTCGGGAGCGGAGGCGGTGGAAGCGGTGGGGGAGGGTCCGGGGGAGGAGGATCTCAGCCTGTTCTTACTCAATCTTCTTCCCTC TCCGCCTCACCCGGGGCCTCCGCCTCACTGACCTGCACTCTGCGATCAGGCATCAACGTTGGGCCTTATAGAATCTACTGGTACCAGCAAAAGCCTGGATCACCGCCCCAGTACCTGCTGAACTATAAATCAGACTCAGACAAGCAGCAGGGCTCCGGCGTGCCGAGTCGATTTAGCGGGAGCAAGGACGCGTCTGCTAATGCCGGCGTGCTTCTCATCAGCGGGCTCCGCAGTGAGGATGAGGCAGATTACTACTGGC ATTTGGCATAGCAGTGCAGCCGTATTTGGCGGAGGAACACAGCTGACTGTCCTCTCTCGCGCCGCCGCTCCGACCACCACCCCTGCACCACGCCCACCTACTCCTGCGCCAACCATTGCCAGCCAGCCTCTCTCTCTCCGACCCGAGGCCTGTAGACCTGCCGCTGGCGGTGCAGTTCATACTCGGGGTCTCGATTTCGCCTGCGACATCTACATCTGGGCACCACTGGCTGGCACTTGTGGCGTTTTGCTCCTGTCCCTG GTGATCACTCTCTACTGTAATAAGAGGGGGAGGAAGAAACTCCTGTATATTTTCAAACAACCCTTTATGCGCCCTGTCCAAACCACCCAGGAAGAAGATGGATGTAGTTGCAGATTCCCAGAAGAAGAGGAGGGTGGGTGTGAACTTAGGGTGAAGTTTAGTCGCAGTGCCGACGCTCCCGCTTACCAACAGGGTCAGAACCAACTCTACAATGAGCTGAATCTGGGGAGGCGCGAAGAATACGACGTTCTGGATAAAAAAA GACGCGGCCGCGACCCCGAGATGGGCGGGAAACCGCGGAGAAAGAACCCACAGGAAGGATTGTACAATGAGCTCCAGAAAGATAAGATGGCAGAAGGCCTACTCCGAGATCGGCATGAAGGGGGAGCGAAGGCGCGGGAAAGGACACGATGGGCTGTACCAGGGTCTTTCAACCGCGACAAAGGACACCTATGATGCTCTCCATATGCAGGCCCTCCCGCCACGCGGAAGTGGAGCAACTAATTTCAATAGCCTTCTGAAA GCTGGCGATGTTGAGGAAAATCCTGGGCCGATGCGCATCTCCAAGCCCCATCTGAGGAGCATCAGCATCCAGTGCTACCTGTGTCTGCTGCTCAACAGCCACTTCCTGACGGAAGCAGGCATTCATGTCTTTATCCTGGGATGCTTTTCTGCCGGCCTGCCAAAGACAGAAGCAAACTGGGTTAACGTTATCAGTGATCTGAAAAAAATCGAGGACCTGATCCAGTCCATGCATATTGAGCGCTACGCTGTATACAGAGTCCGA CGTCCACCCATCATGCAAGGTGACCGCTATGAAGTGTTTCCTGCTGGAACTGCAGGTTATCAGCTTGGAAAGTGGCGACGCTTCCATTCACGATACGGTGGAGAACTTGATAATCCTTGCGAATAATAGTCTGAGCAGCAACGGCAACGTTACTGAAAGCGGGTGCAAAGAATGTGAAGAGCTCGAAGAGAAAAACATCAAAGAATTTTTGCAGTCTTTCGTGCATATTGTTCAGATGTTTATTAACACCAGTGGATCAGGAGCAACT AACTTCTCTCTTCTTAAGCAAGCTGGCGATGTAGAGGAAAACCCTGGGCCTATGGCCCTCTTGCTCGCACTGTCCCTCCTGGTCCTGTGGACATCACCCGCCCCCACCCTGTCCGGCACGAATGACGCAGAAGACTGCTGCCTGTCTGTCACGCAGAAACCCATCCCCGGCTATATAGTGCGGAACTTCCATTACCTGCTGATCAAGGACGGATGTAGGGTGCCAGCCGTCGTCTTCACCACCCTGCGAGGGCGCCAGC TTGGCCTCCTCCTGACCAGCCCTGGGTGGAGCGGATCATTCAACGCTTGCAGCGCACCTCAGCAAAAATGAAAAGAAGAAGTAGTTGA (SEQ ID NO: 61). In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 85% sequence identity to SEQ ID NO:61. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 90% sequence identity to SEQ ID NO:61. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 95% sequence identity to SEQ ID NO:61. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 96% sequence identity to SEQ ID NO:61. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 97% sequence identity to SEQ ID NO:61. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 98% sequence identity to SEQ ID NO:61. In some cases, the isolated nucleic acid molecule comprises a sequence comprising about 99% sequence identity to SEQ ID NO:61. In some cases, the isolated nucleic acid molecule includes SEQ ID NO:61. In some cases, the isolated nucleic acid molecule consists of SEQ ID NO:61.

In some embodiments, the isolated nucleic acid molecule comprises a polynucleotide encoding a CAR that specifically binds mesothelin, a polynucleotide encoding sushi 15, and a polynucleotide encoding CCL19. 일부 경우에, 단리된 핵산 분자는 MDWTWRILFLVAAATGAHSQVQLQQSGPGLVTPSQTLSLTCAISGDSVSSNSATWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRMSINPDTSKNQFSLQLNSVTPEDTAVYYCARGMMTYYYGMDVWGQGTTVTVSS GILGSGGGGSGGGGSGGGGS QPVLTQSSSLSASPGASASLTCTLRSGINVGPYRIYWYQQKPGSPPQYLLNYKSDSDKQQGSGVPSRFSGSKDASANAGVLLISGLRSEDEADYYCMIWHSSAAVFGGGTQLTVLS RAAA IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVAFIIFWVRSK RSRLLHSDYMNMTPRRPGPTRKHYQPYAPPRDFAAYRSRVKFSRSADAPAYQQGQNQLYNELNLGRREEYDVLDKRRGRDPEMGGKPRRKNPQEGLYNELQKDKMAEAYSEIGMKGERRRGKGHDGLYQGLSTATKDTYDALHMQALPPRGSGATNFSLLKQAGDVEENPGPMAPRRARGCRTLGLPALLLLLLLRPPATRGITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIR SGGSGGGGSGGGSGGGGSLQ NWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGSGATNFSLLKQAGDVEENPGPMALLLALSLLVLWTSPAPTLSGTNDAEDCCLSVTQKPIPGYIVRNFHYLLIKDGCRVPAVVFTTLRGRQLCAPPDQPWVERIIQRLQRTSAKMKRRSS (서열번호 57)(또한 본원에서 ssVH-P4-CD28힌지-TM-CD28cyto-CD3z-P2A-IL15Rasp-IL15Ra(sushi)-20aa링커 About 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% for -IL15-P2A-endospCCL19 or SSVHsp-P4-CD28-sushi15-CCL19) Encodes a polypeptide containing sequence identity. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 85% sequence identity to SEQ ID NO:57. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:57. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:57. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:57. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 97% sequence identity to SEQ ID NO:57. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:57. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:57. In some cases, the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO:57. In some cases, the isolated nucleic acid molecule encodes a polypeptide consisting of SEQ ID NO:57.

vector

In some embodiments, the one or more vectors comprise a polynucleotide encoding CAR, a polynucleotide encoding IL-15, and optionally a polynucleotide encoding CCL19. In some embodiments, the vector (e.g., expression vector) is a chimeric antigen comprising an antibody that specifically recognizes human mesothelin, a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular region, and a CD3ζ intracellular region. A polynucleotide encoding a receptor (CAR); A polynucleotide encoding IL-15; and optionally a nucleic acid molecule comprising a polynucleotide encoding CCL19. See Figures 1A and 1B.

In some embodiments, the polynucleotide encoding CAR and the polynucleotide encoding IL-15 are arranged from 5' end to 3' end in a vector (e.g., an expression vector) as follows:

A polynucleotide encoding CAR - a polynucleotide encoding IL-15; or

Polynucleotide encoding IL-15 - Polynucleotide encoding CAR.

In some embodiments, the polynucleotide encoding CAR, the polynucleotide encoding IL-15, and the polynucleotide encoding CCL19 are arranged from 5' end to 3' end in a vector (e.g., an expression vector) as follows: :

a polynucleotide encoding CAR - a polynucleotide encoding IL-15 - a polynucleotide encoding CCL19;

a polynucleotide encoding CAR - a polynucleotide encoding CCL19 - a polynucleotide encoding IL-15;

A polynucleotide encoding IL-15 - a polynucleotide encoding CAR - a polynucleotide encoding CCL19;

A polynucleotide encoding CCL19 - a polynucleotide encoding CAR - a polynucleotide encoding IL-15;

a polynucleotide encoding IL-15 - a polynucleotide encoding CCL19 - a polynucleotide encoding CAR; or

Polynucleotide encoding CCL19 - Polynucleotide encoding IL-15 - Polynucleotide encoding CAR.

In some embodiments, the first vector (e.g., first expression vector) comprises a polynucleotide encoding CAR and the second vector (e.g., second expression vector) comprises a polynucleotide encoding IL-15. .

In some embodiments, the first vector (e.g., a first expression vector) comprises a polynucleotide encoding CAR, and the second vector (e.g., a second expression vector) comprises a polynucleotide encoding IL-15 and CCL19. A polynucleotide encoding IL-15, wherein the polynucleotide encoding IL-15 and the polynucleotide encoding CCL19 optionally encode IL-15 from the 5' end to the 3' end in a second vector (e.g., a second expression vector). A polynucleotide encoding - a polynucleotide encoding CCL19 or a polynucleotide encoding CCL19 - a polynucleotide encoding IL-15.

In a further embodiment, the first vector (e.g., first expression vector) comprises a polynucleotide encoding CAR and a polynucleotide encoding IL-15 or a polynucleotide encoding CCL19, and the second vector (e.g., 2 expression vector) comprises a polynucleotide encoding IL-15 or a polynucleotide encoding CCL19 that is not included in the first vector. In some embodiments, the first vector (e.g., a first expression vector) comprises a polynucleotide encoding CAR and a polynucleotide encoding IL-15, and the second vector (e.g., a second expression vector) comprises CCL19. Contains a polynucleotide that encodes In other embodiments, the first vector (e.g., a first expression vector) comprises a polynucleotide encoding CAR and a polynucleotide encoding CCL19, and the second vector (e.g., a second expression vector) comprises IL-15. Contains a polynucleotide that encodes

In a further embodiment, the first vector (e.g., first expression vector) comprises a polynucleotide encoding CAR, and the second vector (e.g., second expression vector) comprises a polynucleotide encoding IL-15, and , the third vector (eg, third expression vector) comprises a polynucleotide encoding CCL19.

In some embodiments, vectors (e.g., expression vectors) described herein include a polynucleotide encoding mIL-15/Ra and a polynucleotide encoding CCL19. In some cases, the vector (e.g., expression vector) is about 80%, 85%, 90%, 95%, 96%, 97% for SEQ ID NO: 14 (also referred to herein as mIL15/Ra-P2A-CCL19) , comprising a nucleic acid sequence encoding a polypeptide comprising 98%, 99%, or 100% sequence identity. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 85% sequence identity to SEQ ID NO:14. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:14. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:14. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 96% sequence identity to SEQ ID NO:14. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:14. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:14. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:14. In some cases, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO:14. In some cases, the nucleic acid sequence encodes a polypeptide consisting of SEQ ID NO:14.

In some embodiments, vectors (e.g., expression vectors) described herein include a polynucleotide encoding sushi15 and a polynucleotide encoding CCL19. In some cases, the vector (e.g., expression vector) is about 80%, 85%, 90%, 95%, 96%, 97%, 98%, It includes a nucleic acid sequence encoding a polypeptide comprising 99%, or 100% sequence identity. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 85% sequence identity to SEQ ID NO:31. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 90% sequence identity to SEQ ID NO:31. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:31. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:31. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 97% sequence identity to SEQ ID NO:31. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:31. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:31. In some cases, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO:31. In some cases, the nucleic acid sequence encodes a polypeptide consisting of SEQ ID NO:31.

In some embodiments, vectors (e.g., expression vectors) described herein include a polynucleotide encoding a CAR that specifically binds mesothelin and a polynucleotide encoding mIL-15/Ra. In some cases, the vector (e.g., expression vector) is about 80% for SEQ ID NO: 34 (also referred to herein as ssVH-P4-CAR-P2A-IL2sp-mIL15/Ra or P4-BB-mIL15/Ra), Includes a nucleic acid sequence encoding a polypeptide comprising 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 85% sequence identity to SEQ ID NO:34. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:34. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:34. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:34. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:34. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:34. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:34. In some cases, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO:34. In some cases, the nucleic acid sequence encodes a polypeptide consisting of SEQ ID NO:34.

In some embodiments, the vector comprising a nucleic acid sequence encoding ssVH-P4-CAR-P2A-IL2sp-mIL15/Ra or P4-BB-mIL15/Ra has about 80%, 85%, 90% relative to SEQ ID NO:37. , includes sequences comprising 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the nucleic acid sequence includes a sequence comprising about 85% sequence identity to SEQ ID NO:37. In some cases, the nucleic acid sequence includes a sequence comprising about 90% sequence identity to SEQ ID NO:37. In some cases, the nucleic acid sequence includes a sequence comprising about 95% sequence identity to SEQ ID NO:37. In some cases, the nucleic acid sequence includes a sequence comprising about 96% sequence identity to SEQ ID NO:37. In some cases, the nucleic acid sequence includes a sequence comprising about 97% sequence identity to SEQ ID NO:37. In some cases, the nucleic acid sequence includes a sequence comprising about 98% sequence identity to SEQ ID NO:37. In some cases, the nucleic acid sequence includes a sequence comprising about 99% sequence identity to SEQ ID NO:37. In some cases, the nucleic acid sequence includes SEQ ID NO:37. In some cases, the nucleic acid sequence consists of SEQ ID NO:37.

In some embodiments, vectors (e.g., expression vectors) described herein include a polynucleotide encoding a CAR that specifically binds mesothelin and a polynucleotide encoding sushi15. In some cases, the vector (e.g., expression vector) has about 80%, 85%, 90%, Includes a nucleic acid sequence encoding a polypeptide comprising 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 85% sequence identity to SEQ ID NO:35. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 90% sequence identity to SEQ ID NO:35. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:35. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:35. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:35. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:35. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:35. In some cases, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO:35. In some cases, the nucleic acid sequence encodes a polypeptide consisting of SEQ ID NO:35.

In some embodiments, the vector comprising a nucleic acid sequence encoding ssVH-P4-CAR-P2A-sushi15 or P4-BB-sushi15 has about 80%, 85%, 90%, 95%, 96% relative to SEQ ID NO: 38. , includes sequences comprising 97%, 98%, 99%, or 100% sequence identity. In some cases, the nucleic acid sequence includes a sequence comprising about 85% sequence identity to SEQ ID NO:38. In some cases, the nucleic acid sequence includes a sequence comprising about 90% sequence identity to SEQ ID NO:38. In some cases, the nucleic acid sequence includes a sequence comprising about 95% sequence identity to SEQ ID NO:38. In some cases, the nucleic acid sequence includes a sequence comprising about 96% sequence identity to SEQ ID NO:38. In some cases, the nucleic acid sequence includes a sequence comprising about 97% sequence identity to SEQ ID NO:38. In some cases, the nucleic acid sequence includes a sequence comprising about 98% sequence identity to SEQ ID NO:38. In some cases, the nucleic acid sequence includes a sequence comprising about 99% sequence identity to SEQ ID NO:38. In some cases, the nucleic acid sequence includes SEQ ID NO:38. In some cases, the nucleic acid sequence consists of SEQ ID NO:38.

In some embodiments, vectors (e.g., expression vectors) described herein include a polynucleotide encoding a CAR that specifically binds mesothelin, a polynucleotide encoding mIL-15/Ra, and a polynucleotide encoding CCL19. do. In some cases, the vector (e.g., expression vector) is for SEQ ID NO: 36 (also referred to herein as ssVH-P4-CAR-P2A-IL2sp-mIL15/Ra-P2A-CCL19 or P4-BB-mIL15-CCL19) and a nucleic acid sequence encoding a polypeptide comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 85% sequence identity to SEQ ID NO:36. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:36. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:36. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:36. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:36. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:36. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:36. In some cases, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO:36. In some cases, the nucleic acid sequence encodes a polypeptide consisting of SEQ ID NO:36.

In some embodiments, the vector comprising a nucleic acid sequence encoding ssVH-P4-CAR-P2A-IL2sp-mIL15/Ra-P2A-CCL19 or P4-BB-mIL15-CCL19 has about 80% for SEQ ID NO:39, 85 %, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the nucleic acid sequence includes a sequence comprising about 85% sequence identity to SEQ ID NO:39. In some cases, the nucleic acid sequence includes a sequence comprising about 90% sequence identity to SEQ ID NO:39. In some cases, the nucleic acid sequence includes a sequence comprising about 95% sequence identity to SEQ ID NO:39. In some cases, the nucleic acid sequence includes a sequence comprising about 96% sequence identity to SEQ ID NO:39. In some cases, the nucleic acid sequence includes a sequence comprising about 97% sequence identity to SEQ ID NO:39. In some cases, the nucleic acid sequence includes a sequence comprising about 98% sequence identity to SEQ ID NO:39. In some cases, the nucleic acid sequence includes a sequence comprising about 99% sequence identity to SEQ ID NO:39. In some cases, the nucleic acid sequence includes SEQ ID NO:39. In some cases, the nucleic acid sequence consists of SEQ ID NO:39.

In some embodiments, vectors (e.g., expression vectors) described herein include a polynucleotide encoding a CAR that specifically binds mesothelin, a polynucleotide encoding sushi15, and a polynucleotide encoding CCL19. In some cases, the vector (e.g., expression vector) is about 80% for SEQ ID NO: 15 (also referred to herein as ssVH-P4-CAR-P2A-sushi15-P2A-CCL19 or P4-BB-sushi15-CCL19), Includes a nucleic acid sequence encoding a polypeptide comprising 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 85% sequence identity to SEQ ID NO:15. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 90% sequence identity to SEQ ID NO:15. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:15. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:15. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:15. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:15. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:15. In some cases, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO:15. In some cases, the nucleic acid sequence encodes a polypeptide consisting of SEQ ID NO:15.

In some embodiments, the vector comprising a nucleic acid sequence encoding ssVH-P4-CAR-P2A-sushi15-P2A-CCL19 or P4-BB-sushi15-CCL19 has about 80%, 85%, 90% relative to SEQ ID NO:40. , includes sequences comprising 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the nucleic acid sequence includes a sequence comprising about 85% sequence identity to SEQ ID NO:40. In some cases, the nucleic acid sequence includes a sequence comprising about 90% sequence identity to SEQ ID NO:40. In some cases, the nucleic acid sequence includes a sequence comprising about 95% sequence identity to SEQ ID NO:40. In some cases, the nucleic acid sequence includes a sequence comprising about 96% sequence identity to SEQ ID NO:40. In some cases, the nucleic acid sequence includes a sequence comprising about 97% sequence identity to SEQ ID NO:40. In some cases, the nucleic acid sequence includes a sequence comprising about 98% sequence identity to SEQ ID NO:40. In some cases, the nucleic acid sequence includes a sequence comprising about 99% sequence identity to SEQ ID NO:40. In some cases, the nucleic acid sequence includes SEQ ID NO:40. In some cases, the nucleic acid sequence consists of SEQ ID NO:40.

In some embodiments, vectors (e.g., expression vectors) described herein include a polynucleotide encoding a CAR that specifically binds mesothelin and a polynucleotide encoding mIL15/Ra-LSP. In some cases, the vector (e.g., expression vector) is SEQ ID NO: 53 (also herein referred to as ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15propeptide-mIL15/Ra or SSVHsp-P4-BB-mIL15 A nucleic acid sequence encoding a polypeptide comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to (referred to as /Ra-LSP) Includes. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 85% sequence identity to SEQ ID NO:53. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:53. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:53. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:53. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:53. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:53. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:53. In some cases, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO:53. In some cases, the nucleic acid sequence encodes a polypeptide consisting of SEQ ID NO:53.

In some embodiments, the vector comprising a nucleic acid sequence encoding ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15propeptide-mIL15/Ra or SSVHsp-P4-BB-mIL15/Ra-LSP and sequences comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:58. In some cases, the nucleic acid sequence includes a sequence comprising about 85% sequence identity to SEQ ID NO:58. In some cases, the nucleic acid sequence includes a sequence comprising about 90% sequence identity to SEQ ID NO:58. In some cases, the nucleic acid sequence includes a sequence comprising about 95% sequence identity to SEQ ID NO:58. In some cases, the nucleic acid sequence includes a sequence comprising about 96% sequence identity to SEQ ID NO:58. In some cases, the nucleic acid sequence includes a sequence comprising about 97% sequence identity to SEQ ID NO:58. In some cases, the nucleic acid sequence includes a sequence comprising about 98% sequence identity to SEQ ID NO:58. In some cases, the nucleic acid sequence includes a sequence comprising about 99% sequence identity to SEQ ID NO:58. In some cases, the nucleic acid sequence includes SEQ ID NO:58. In some cases, the nucleic acid sequence consists of SEQ ID NO:58.

In some embodiments, vectors (e.g., expression vectors) described herein include a polynucleotide encoding a CAR that specifically binds mesothelin and a polynucleotide encoding sIL15-LSP. In some cases, the vector (e.g., expression vector) is SEQ ID NO: 54 (also herein referred to as ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15propeptide-IL15 or SSVHsp-P4-BB-sIL15-LSP (referred to as ). In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 85% sequence identity to SEQ ID NO:54. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 90% sequence identity to SEQ ID NO:54. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:54. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:54. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:54. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:54. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:54. In some cases, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO:54. In some cases, the nucleic acid sequence encodes a polypeptide consisting of SEQ ID NO:54.

In some embodiments, the vector comprising a nucleic acid sequence encoding ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15propeptide-IL15 or SSVHsp-P4-BB-sIL15-LSP is set to SEQ ID NO:59. and a sequence comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity. In some cases, the nucleic acid sequence includes a sequence comprising about 85% sequence identity to SEQ ID NO:59. In some cases, the nucleic acid sequence includes a sequence comprising about 90% sequence identity to SEQ ID NO:59. In some cases, the nucleic acid sequence includes a sequence comprising about 95% sequence identity to SEQ ID NO:59. In some cases, the nucleic acid sequence includes a sequence comprising about 96% sequence identity to SEQ ID NO:59. In some cases, the nucleic acid sequence includes a sequence comprising about 97% sequence identity to SEQ ID NO:59. In some cases, the nucleic acid sequence includes a sequence comprising about 98% sequence identity to SEQ ID NO:59. In some cases, the nucleic acid sequence includes a sequence comprising about 99% sequence identity to SEQ ID NO:59. In some cases, the nucleic acid sequence includes SEQ ID NO:59. In some cases, the nucleic acid sequence consists of SEQ ID NO:59.

In some embodiments, vectors (e.g., expression vectors) described herein include a polynucleotide encoding a CAR that specifically binds mesothelin, a polynucleotide encoding mIL15/Ra-LSP, and a polynucleotide encoding CCL19. do. In some cases, the vector (e.g., an expression vector) is SEQ ID NO: 55 (also herein referred to as ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15propeptide-IL15-P2A-endospCCL19 or ssVHsp-P4-BB -mIL15/Ra-LSP-CCL19) encoding a polypeptide comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to It contains a nucleic acid sequence. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 85% sequence identity to SEQ ID NO:55. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:55. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:55. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 96% sequence identity to SEQ ID NO:55. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:55. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 98% sequence identity to SEQ ID NO:55. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:55. In some cases, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO:55. In some cases, the nucleic acid sequence encodes a polypeptide consisting of SEQ ID NO:55.

In some embodiments, a nucleic acid sequence encoding ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15propeptide-IL15-P2A-endospCCL19 or ssVHsp-P4-BB-mIL15/Ra-LSP-CCL19 The containing vector contains a sequence comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:60. In some cases, the nucleic acid sequence includes a sequence comprising about 85% sequence identity to SEQ ID NO:60. In some cases, the nucleic acid sequence includes a sequence comprising about 90% sequence identity to SEQ ID NO:60. In some cases, the nucleic acid sequence includes a sequence comprising about 95% sequence identity to SEQ ID NO:60. In some cases, the nucleic acid sequence includes a sequence comprising about 96% sequence identity to SEQ ID NO:60. In some cases, the nucleic acid sequence includes a sequence comprising about 97% sequence identity to SEQ ID NO:60. In some cases, the nucleic acid sequence includes a sequence comprising about 98% sequence identity to SEQ ID NO:60. In some cases, the nucleic acid sequence includes a sequence comprising about 99% sequence identity to SEQ ID NO:60. In some cases, the nucleic acid sequence includes SEQ ID NO:60. In some cases, the nucleic acid sequence consists of SEQ ID NO:60.

In some embodiments, vectors (e.g., expression vectors) described herein include a polynucleotide encoding a CAR that specifically binds mesothelin, a polynucleotide encoding sIL15-LSP, and a polynucleotide encoding CCL19. In some cases, the vector (e.g., expression vector) is SEQ ID NO: 56 (also herein referred to as ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15propeptide-IL15-P2A-endospCCL19 or ssVHsp-P4-BB A nucleic acid encoding a polypeptide comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to (referred to as -sIL15-LSP-CCL19) Includes sequence. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 85% sequence identity to SEQ ID NO:56. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 90% sequence identity to SEQ ID NO:56. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:56. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:56. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:56. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:56. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:56. In some cases, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO:56. In some cases, the nucleic acid sequence encodes a polypeptide consisting of SEQ ID NO:56.

In some embodiments, comprising a nucleic acid sequence encoding ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15propeptide-IL15-P2A-endospCCL19 or ssVHsp-P4-BB-sIL15-LSP-CCL19 The vector contains a sequence comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to SEQ ID NO:61. In some cases, the nucleic acid sequence includes a sequence comprising about 85% sequence identity to SEQ ID NO:61. In some cases, the nucleic acid sequence includes a sequence comprising about 90% sequence identity to SEQ ID NO:61. In some cases, the nucleic acid sequence includes a sequence comprising about 95% sequence identity to SEQ ID NO:61. In some cases, the nucleic acid sequence includes a sequence comprising about 96% sequence identity to SEQ ID NO:61. In some cases, the nucleic acid sequence includes a sequence comprising about 97% sequence identity to SEQ ID NO:61. In some cases, the nucleic acid sequence includes a sequence comprising about 98% sequence identity to SEQ ID NO:61. In some cases, the nucleic acid sequence includes a sequence comprising about 99% sequence identity to SEQ ID NO:61. In some cases, the nucleic acid sequence includes SEQ ID NO:61. In some cases, the nucleic acid sequence consists of SEQ ID NO:61.

In some embodiments, vectors (e.g., expression vectors) described herein include a polynucleotide encoding a CAR that specifically binds mesothelin, a polynucleotide encoding sushi15, and a polynucleotide encoding CCL19. In some cases, the vector (e.g., an expression vector) is SEQ ID NO: 57 (also herein referred to as ssVH-P4-CD28hinge-TM-CD28cyto-CD3z-P2A-IL15Rasp-IL15Ra(sushi)-20aa linker-IL15-P2A-endospCCL19 or a polypeptide comprising about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to (referred to as ssVHsp-P4-CD28-sushi15-CCL19) It contains a nucleic acid sequence that encodes. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 85% sequence identity to SEQ ID NO:57. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 90% sequence identity to SEQ ID NO:57. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 95% sequence identity to SEQ ID NO:57. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 96% sequence identity to SEQ ID NO:57. In some cases, the nucleic acid sequence encodes a polypeptide comprising approximately 97% sequence identity to SEQ ID NO:57. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 98% sequence identity to SEQ ID NO:57. In some cases, the nucleic acid sequence encodes a polypeptide comprising about 99% sequence identity to SEQ ID NO:57. In some cases, the nucleic acid sequence encodes a polypeptide comprising SEQ ID NO:57. In some cases, the nucleic acid sequence encodes a polypeptide consisting of SEQ ID NO:57.

The vector (e.g., expression vector) of the present invention may include one or more naturally derived nucleic acids or artificially synthesized nucleic acids, and may be appropriately selected depending on the type of cell into which the vector (e.g., expression vector) of the present invention will be introduced. can be selected. Their sequence information can be appropriately obtained by searching publicly known literature or databases, such as NCBI (www.ncbi.nlm.nih.gov/guide/).

The vector of the present invention is an expression vector that can be introduced into an immune cell or its precursor cell by contacting the vector with a cell, and a predetermined protein (polypeptide) encoded therein is expressed within the immune cell to produce the modified immune cell of the present invention. It can be. The expression vector of the present invention is not particularly limited by any embodiment. Those skilled in the art can design and produce expression vectors that allow expression of desired proteins (polypeptides) in immune cells. Examples of expression vectors of the invention comprising a polynucleotide encoding a cell surface molecule that specifically recognizes human mesothelin, a polynucleotide encoding IL-15 and optionally a polynucleotide encoding CCL19 are examples of expression vectors of the invention for use in immune cells of the invention. It may include any of the expression vectors for producing.

The type of expression vector of the present invention may be linear or circular, may be a non-viral vector such as a plasmid, may be a viral vector, or may be a transposon-based vector. Such vectors may contain control sequences, such as promoters or terminators, or selective marker sequences, such as drug resistance genes or reporter genes. The polynucleotide encoding IL-15 and the polynucleotide encoding CCL19 can be operably arranged downstream of the promoter sequence such that each polynucleotide can be efficiently transcribed.

Examples of promoters may include: viral-derived promoters such as the retroviral LTR promoter, SV40 early promoter, cytomegalovirus promoter, and herpes simplex virus thymidine kinase promoter; and mammalian-derived promoters, such as phosphoglycerate kinase (PGK) promoter, Xist promoter, β-actin promoter, and RNA polymerase II promoter. In some embodiments, the promoter may preferably comprise a retroviral LTR promoter. The retroviral LTR promoter is: May include CCAATCAGTTCGCTTCTCGCTTCTGTTCGCGCGCTTCTGCTCCCCGAGCTCAATAAAAGAGCCCACAACCCCTCACTCGGCGCGCCAGTCCTCCGATTGACTGAGTCGCCCGGGTACCCGTGTATCCAATAAACCCTCTTGCAGTTGCATCCGACTTGTGGTCTCGCTGTTCCTTGGGAGGGTCTCCTCTGAGTGATTGACTACCCGTCAGCGGGGGTCTTTCA. Alternatively, a tetracycline-responsive promoter induced by tetracycline, an Mx1 promoter induced by interferon, etc. can be used. The use of a promoter induced by a specific substance in the expression vector of the present invention can be used in the process of treating cancer, for example, when immune cells containing the vector of the present invention are used as a pharmaceutical composition for use in the treatment of cancer. thus allowing control of IL-15 expression and optionally induction of CCL19 expression.

Examples of viral vectors may include retroviral vectors, lentiviral vectors, adenoviral vectors, and adeno-associated viral vectors, preferably retroviral vectors, preferably gamma retroviral vectors, more preferably pMSGV vectors. (Tamada k et al., Clin Cancer Res 18: 6436-6445 (2002)), pMSCV vector (manufactured by Takara Bio Inc.), or pSFG vector. The use of retroviral vectors allows long-term, stable expression of the transgene because the transgene is integrated into the genome of the host cell.

One or more assays can be used to confirm the presence of expression vectors of the invention in immune cells. Exemplary assays may include flow cytometry, Northern blotting, Southern blotting, PCR, such as RT-PCR, ELISA, or Western blotting, to screen for expression of CAR by engineered immune cells. In some embodiments, the expression vector contains a marker gene (e.g., a fluorescent protein, such as green fluorescent protein (GFP), red fluorescent protein (RFP), for detecting expression of CAR, IL-15, and/or CCL19 by immune cells. or encoding yellow fluorescent protein (YFP)).

Immune cells and production methods

In certain embodiments, the immune cells described herein are modified to express IL-15 and, optionally, CCL19, cell surface molecules that specifically recognize mesothelin (e.g., human mesothelin) (Figures 1C and 1D). Exemplary immune cells include lymphoid cells such as T cells, natural killer cells (NK cells) and B cells, antigen presenting cells such as monocytes, macrophages, dendritic cells or granulocytes such as neutrophils, eosinophils, basophils or May contain mast cells. Immune cells may be derived from stem cells, such as induced pluripotent stem cells (iPSC), embryonic stem cells (ESC), ESC-derived hematopoietic progenitor cells (HPC), or hematopoietic endothelial cells (HEC). Immune cells may include T cells derived from mammals, such as humans, dogs, cats, pigs or mice, preferably T cells derived from or isolated from humans. Immune cells (e.g., T cells) can be obtained through culture, such as ex vivo culture, or harvested directly from mammals. Immune cells are not limited as long as the cells participate in immune responses and can express cell surface molecules that specifically recognize mesothelin (e.g., human mesothelin), express IL-15, and optionally express CCL19. No. Immune cells may be autologous cells harvested from a subject in need of them for subsequent treatment. Immune cells may also be allogeneic or syngeneic to the subject in need thereof.

In some embodiments, the immune cells are further modified to impair or improve an inflammatory or immune response in the subject due to administration of the immune cells in the subject. In some cases, the inflammatory or immune response is graft versus host disease (GvHD), and immune cells are further modified to damage or improve GvHD.

In some cases, immune cells are modified to impair or eliminate expression of the alpha and/or beta chains of the T-cell receptor (TCR). In some cases, immune cells are modified to impair or eliminate expression of the alpha and beta chains of TCRs (TCRα/β). In some cases, immune cells do not express the alpha and beta chains of the TCR (TCRα/β). In some cases, one or more of the additional CD3 domains (e.g., CD3 delta, CD3 gamma, or CD3 epsilon) are modified to impair TCRα/β function.

In some cases, immune cells express the gamma delta T cell receptor (gdTCR or γδTCR). γδT cells are a subgroup of T cells and account for approximately 0.5-5% of all T-lymphocytes. Under cancer environments, γδT cells have been shown to exert both pro- and anti-tumor activities, based on the combined subgroups of γ and δ receptors presented on the surface of the cells. For example, γδT cells expressing a TCR containing γ-chain variable region 9 (Vγ9) and δ-chain variable region 2 (Vδ2), also referred to as Vγ9Vδ2 T cells, are capable of promoting, but not limited to, cancer cell proliferation, angiogenesis, It exerts anti-tumor activity including inhibition of lymphangiogenesis and increase in cancer cell apoptosis. Examples of γTCR include Vγ1TCR, Vγ2TCR, Vγ3TCR, Vγ4TCR, Vγ5TCR, Vγ6TCR, Vγ7TCR, Vγ8TCR and Vγ9TCR, and examples of δTCR include Vδ1TCR, Vδ2TCR, Vδ3TCR, Vδ4TCR, Vδ5TCR, Vδ6TCR, Vδ7TCR, Vδ8TCR and Vδ9TCR. Includes. Combinations of specific γTCR and δTCR are not limited, but for example Vγ3Vδ1TCR, Vγ4Vδ1TCR, Vγ9Vδ1TCR and Vγ9Vδ2TCR are contemplated. In some cases, the immune cells express Vγ9 TCR, Vδ2 TCR, or Vγ9Vδ2 TCR.

In some embodiments, the immune cells described herein that express the gamma delta T cell receptor (gdTCR or γδTCR) are produced from induced pluripotent stem cells (iPSCs). In some cases, iPSCs are established from cells other than αβT cells. In some cases, iPSCs are further modified by inserting the Vγ9Vδ2 gene into the genome of the iPSCs prior to differentiation into γδTCR cells. In some cases, the Vγ9Vδ2 gene comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:44. Encodes a polypeptide. In some cases, the Vγ9Vδ2 gene encodes a polypeptide consisting of SEQ ID NO:44.

In some embodiments, the γδTCR cells disclosed herein express a CAR that specifically recognizes mesothelin and are modified to express IL-15. In some cases, γδTCR cells are further modified to express CCL19. In some cases, γδTCR cells are differentiated from iPSCs modified with the Vγ9Vδ2 gene. In some cases, the γδTCR cell comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity to SEQ ID NO:44. Differentiated from iPSCs modified with the Vγ9Vδ2 gene encoding the polypeptide. In some cases, γδTCR cells are differentiated from iPSCs modified with the Vγ9Vδ2 gene encoding the polypeptide consisting of SEQ ID NO:44.

In certain embodiments, disclosed herein are populations of immune cells modified to express CAR and IL-15 that specifically recognize mesothelin. In some cases, populations of immune cells additionally express CCL19. In some embodiments, the population of immune cells comprises modified T cells (e.g., expanded ex vivo or harvested from a mammal) that express a CAR that specifically recognizes mesothelin, IL-15, and optionally CCL19. The population of immune cells may comprise about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or more of modified T cells. The population of immune cells may comprise about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 95% modified T cells. The population of immune cells may comprise a substantially pure population of modified T cells. Exemplary T cells include alpha-beta T cells, gamma-delta T cells, CD8 ⁺ T cells, CD4 ⁺ T cells, tumor-infiltrating T cells, memory T cells, naïve T cells, and natural killer T (NKT) cells. You can.

In some embodiments, the population of immune cells modified to express CAR, IL-15, and optionally CCL19 that specifically recognize mesothelin is about 30%, 25%, 20%, 15%, 10%, 5%, or Contains fewer contaminating cells than less contaminating cells. As used herein, the term “contaminating cells” refers to cells that do not express CAR that specifically recognizes mesothelin, IL-15, and optionally CCL19. Contaminating cells include T cells that specifically recognize mesothelin, but do not express CAR, IL-15, and optionally CCL19, and other types of cells that do not express CAR, IL-15, and optionally CCL19 that specifically recognize mesothelin. May contain immune cells. Contaminating cells may also be non-immune cells from body fluids such as blood or bone marrow fluid, non-immune cells derived from tissues such as spleen tissue, thymus or lymph nodes, or cancer tissues such as primary tumor tissue, metastatic tumor tissue. or may refer to non-immune cells derived from cancerous ascites.

Examples of methods for producing immune cells of the invention may include production methods that introduce polynucleotides encoding cell surface molecules, polynucleotides encoding IL-15, and optionally polynucleotides encoding CCL19 into immune cells. . Production methods are described, for example, in WO2016/056228, WO2017/159736, WO2013/176915, WO2015/120096, WO2016/019300, or Vormittag P et al, Curr Opin Biotechnol 2018; 53: 164-81. Alternative examples include transgenic mammals produced by implanting a cell surface molecule that specifically recognizes mesothelin (e.g., human mesothelin) and/or a vector for the expression of IL-15, and additionally optionally CCL19, into a fertilized egg. Methods for purifying and obtaining immune cells from and, if necessary, vectors for the expression of cell surface molecules that specifically recognize mesothelin (e.g., human mesothelin) and/or IL-15, and further optionally CCL19 It may include a production method of further introducing into immune cells purified and obtained from a transgenic mammal.

When introducing a polynucleotide encoding a cell surface molecule, a polynucleotide encoding IL-15 and optionally a polynucleotide encoding CCL19, or a vector described above, the method includes any of the polynucleotides or vectors that introduce the polynucleotide or vector into an immune cell. It may be a method of. Examples include electroporation (Cytotechnology, 3, 133 (1990)), calcium phosphate method (Japanese Unexamined Patent Application Publication No. 2-227075), and lipofection (Proc. Natl. Acad. Sci. U.S.A., 84, 7413). (1987)) and viral infection methods. Examples of virus infection methods include packaging cells, such as GP2-293 cells (manufactured by Takara Bio Inc.), Plat-GP cells (manufactured by Cosmo Bio Co., Ltd.), PG13 cells (ATCC CRL- 10686) or PA317 cells (ATCC CRL-9078), by transfecting the vector and packaging plasmid to be introduced, producing a recombinant virus, and infecting immune cells with the recombinant virus (e.g., WO2017/159736 reference).

In some embodiments, the method comprises introducing into an immune cell one or more vectors comprising a polynucleotide encoding CAR, a polynucleotide encoding IL-15, and optionally a polynucleotide encoding CCL19. In some embodiments, the method comprises an antibody that specifically recognizes human mesothelin, encoding a chimeric antigen receptor (CAR) comprising a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular region, and a CD3ζ intracellular region. polynucleotide; A polynucleotide encoding IL-15; and optionally introducing a vector (e.g., an expression vector) comprising a nucleic acid molecule comprising a polynucleotide encoding CCL19 into the immune cell. In some embodiments, the method comprises a first vector comprising a polynucleotide encoding CAR (e.g., a first expression vector) and a second vector comprising a polynucleotide encoding IL-15 (e.g., a second expression vector) It includes introducing them into immune cells together or step by step. In some embodiments, the method comprises a first vector (e.g., a first expression vector) comprising a polynucleotide encoding CAR and a second vector comprising a polynucleotide encoding IL-15 and a polynucleotide encoding CCL19 ( For example, introducing a second expression vector) into the immune cells together or step by step. In some embodiments, the method includes a first vector (e.g., a first expression vector) comprising a polynucleotide encoding CAR and a polynucleotide encoding IL-15 or a polynucleotide encoding CCL19, and encoding IL-15. and introducing a second vector (e.g., a second expression vector) containing a polynucleotide encoding CCL19 that is not included in the first vector or a polynucleotide encoding CCL19 together or stepwise into the immune cell. In some embodiments, the method comprises a first vector (e.g., a first expression vector) comprising a polynucleotide encoding CAR and a polynucleotide encoding IL-15, and a second vector comprising a polynucleotide encoding CCL19. and introducing (e.g., a second expression vector) into the immune cells together or stepwise. In some embodiments, the method comprises a first vector (e.g., a first expression vector) comprising a polynucleotide encoding CAR and a polynucleotide encoding CCL19, and a second vector comprising a polynucleotide encoding IL-15. and introducing (e.g., a second expression vector) into the immune cells together or stepwise. In some embodiments, the method comprises a first vector comprising a polynucleotide encoding CAR (e.g., a first expression vector), a second vector comprising a polynucleotide encoding IL-15 (e.g., a second expression vector) , and a third vector (e.g., a third expression vector) comprising a polynucleotide encoding CCL19 together or stepwise into the immune cell.

One or more of a polynucleotide encoding CAR, a polynucleotide encoding IL-15, or a polynucleotide encoding CCL19 may be integrated into the genome of an immune cell. In some embodiments, the polynucleotide encoding CAR, the polynucleotide encoding IL-15, and the polynucleotide encoding CCL19 are not integrated into the genome (e.g., episomally).

How to use

In certain embodiments, disclosed herein are methods of treating mesothelin-expressing cancer. In some embodiments, the method comprises an immune cell described herein that has been modified to express an engineered cell surface molecule that specifically binds mesothelin, interleukin 15 (IL-15) and, optionally, chemokine (C-C motif) ligand 19 (CCL19). It includes administering to a subject in need thereof. In some embodiments, the immune cells are modified to express engineered cell surface molecules comprising a chimeric antigen receptor (CAR) that specifically recognizes mesothelin or a T cell receptor (TCR) that specifically binds mesothelin. . In some embodiments, the immune cell comprises an antibody that specifically recognizes human mesothelin, a CAR comprising a CD8 hinge region, a CD8 transmembrane region, a 4-1BB intracellular region, and a CD3ζ intracellular region; IL-15; and optionally modified to express CCL19.

In some embodiments, the mesothelin-expressing cancer is a solid tumor. In some embodiments, the solid tumor is mesothelioma, colorectal cancer, pancreatic cancer, thymic cancer, bile duct cancer, lung cancer, skin cancer, breast cancer, prostate cancer, urinary bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, gastric cancer, esophageal cancer. Includes bowel cancer, tracheal cancer, bronchial cancer, stomach cancer, esophageal cancer, gallbladder cancer, testicular cancer, ovarian cancer, or bone cancer. In some embodiments, the mesothelin-expressing cancer is ovarian cancer. In some embodiments, the mesothelin-expressing cancer is mesothelioma. In some embodiments, the mesothelin-expressing cancer is gastric cancer. In some embodiments, the mesothelin-expressing cancer is lung cancer (e.g., non-small cell lung cancer (NSCLC), small cell lung cancer (SCLC), lung carcinoid tumor, adenosquamous carcinoma of the lung, large cell neuroendocrine carcinoma, or salivary gland- type lung carcinoma). In some embodiments, the mesothelin-expressing cancer is NSCLC (e.g., adenocarcinoma, squamous cell, large-cell undifferentiated carcinoma, sarcomatoid carcinoma, or adenosquamous carcinoma of the lung).

Mesothelin-expressing cancers may be hematopoietic cancers. The hematopoietic cancer may be B-cell hematopoietic cancer, T-cell hematopoietic cancer, Hodgkin's lymphoma, or non-Hodgkin's lymphoma. Hematopoietic cancers include acute lymphocytic leukemia (ALL), chronic lymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), and mantle cell lymphoma (MCL). , marginal zone lymphoma, Burkitt's lymphoma, or Waldenstrom's macroglobulinemia.

Hematopoietic cancer may be a sarcoma. Sarcomas may include chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, or soft tissue sarcoma.

The mesothelin-expressing cancer may be a metastatic cancer, such as a metastatic solid tumor or a metastatic hematopoietic cancer. The metastatic mesothelin-expressing cancer may be metastatic ovarian cancer, metastatic mesothelioma, metastatic gastric cancer, or metastatic lung cancer (eg, metastatic NSCLC).

The mesothelin-expressing cancer may be a relapsed or refractory cancer, such as a relapsed or refractory solid tumor, or a relapsed or refractory hematopoietic cancer. Relapsed or refractory mesothelin-expressing cancers include relapsed or refractory ovarian cancer, relapsed or refractory mesothelioma, relapsed or refractory gastric cancer, or relapsed or refractory lung cancer (e.g., relapsed or refractory NSCLC). It can be.

In some embodiments, the method further comprises administering an additional therapeutic agent or additional therapeutic regimen to the subject. Additional therapeutic agents may include chemotherapy agents, immunotherapy agents, targeted therapy, radiation therapy, or combinations thereof. Exemplary additional therapeutic agents include alkylating agents such as altretamine, busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, dacarbazine, lomustine, melphalan, oxaliplatin, temozolomide, or thiotepa; Antimetabolites such as 5-fluorouracil (5-FU), 6-mercaptopurine (6-MP), capecitabine, cytarabine, floxuridine, fludarabine, gemcitabine, hydroxyurea, methotrexate or pemetrexed; Anthracyclines such as daunorubicin, doxorubicin, epirubicin or idarubicin; Topoisomerase I inhibitors such as topotecan or irinotecan (CPT-11); Topoisomerase II inhibitors such as etoposide (VP-16), teniposide or mitoxantrone; Mitotic inhibitors such as docetaxel, estramustine, ixabepilone, paclitaxel, vinblastine, vincristine or vinorelbine; or corticosteroids such as prednisone, methylprednisolone, or dexamethasone.

In some embodiments, the additional therapeutic agent comprises a first line therapy. As used herein, “first line therapy” includes primary treatment for a subject with cancer. In some embodiments, the cancer is a primary cancer. In other embodiments, the cancer is metastatic or recurrent cancer. In some embodiments, the first line therapy includes chemotherapy. In other embodiments, the first line treatment includes radiation therapy. Those skilled in the art will readily understand that different first line treatments may be applied to different types of cancer.

In some embodiments, the additional therapeutic agent includes an inhibitor of the poly ADP ribose polymerase (PARP) enzyme. Exemplary PARP inhibitors include olaparib (AZD-2281, Lynparza®, from Astra Zeneca), rucaparib (PF-01367338, Rubraca®, from Clovis Oncology), niraparib (MK-4827, Zejula®) , from Tesaro), talazoparib (BMN-673, from BioMarin Pharmaceutical Inc.), veliparib (ABT-888, from Abb Vie), CK-102 (formerly CEP 9722, from Teva Pharmaceutical Industries Ltd. ), E7016 (from Eisai), iniparib (BSI 201, from Sanofi), and pamiparib (BGB-290, from BeiGene).

In some embodiments, the additional therapeutic agent includes an immune checkpoint inhibitor. In some embodiments, the checkpoint inhibitor includes pembrolizumab, nivolumab, tremelimumab, or ipilimumab. In some embodiments, the checkpoint inhibitor is PD-L1, PD-L2, PD-1, CTLA-4, LAG3, B7-H3, KIR, CD137, PS, TFM3, CD52, CD30, CD20, CD33, CD27, OX40. , GITR, ICOS, BTLA (CD272), CD160, 2B4, LAIR1, TIGHT, LIGHT, DR3, CD226, CD2 or SLAM. The inhibitor may be an antibody or fragment thereof (e.g., a monoclonal antibody, human, humanized, or chimeric antibody), an RNAi molecule, or a small molecule.

In some embodiments, the additional therapeutic agent comprises an antibody such as alemtuzumab, trastuzumab, ibritumomab tiuxetan, brentuximab vedotin, ado-trastuzumab emtansine, or blinatumomab. .

In some embodiments, the additional therapeutic agent comprises a cytokine. Exemplary cytokines include, but are not limited to, IL-1β, IL-6, IL-7, IL-10, IL-12, IL-21, or TNFα.

In some embodiments, the additional therapeutic agent includes a receptor agonist. In some embodiments, the receptor agonist comprises a toll-like receptor (TLR) ligand. In some embodiments, the TLR ligand comprises TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, or TLR9. In some embodiments, the TLR ligand is a synthetic ligand, such as, e.g., Pam3Cys, CFA, MALP2, Pam2Cys, FSL-1, Hib-OMPC, Poly I:C, Poly A:U, AGP, MPL A, RC- 529, MDF2p, CFA or flagellin.

In some embodiments, additional therapeutic agents include fludarabine and cyclophosphamide.

In some embodiments, the additional therapeutic agent includes tisagenlecleucel (Kymria®), axicabtagen ciloleucel (Yekarta®), or brexucabtagen autoleucel (Tecartus®).

In some embodiments, the additional treatment regimen includes surgery.

In some embodiments, the immune cells described herein or the pharmaceutical composition described herein and the additional therapeutic agent are administered simultaneously.

In some embodiments, the immune cells described herein or the pharmaceutical composition described herein and the additional therapeutic agent are administered sequentially. In some embodiments, the immune cells described herein or the pharmaceutical compositions described herein are administered to the subject prior to administration of additional therapeutic agents. In other embodiments, the immune cells described herein or the pharmaceutical compositions described herein are administered to the subject following administration of an additional therapeutic agent.

In some embodiments, the subject is a human.

In some embodiments, methods of producing immune cells expressing a cell surface molecule that specifically recognizes mesothelin (e.g., human mesothelin), IL-15, and optionally CCL19 are also described herein. The method includes introducing a nucleic acid molecule described herein or a vector comprising a nucleic acid molecule into an immune cell, thereby inducing expression of IL-15, a cell surface molecule that specifically recognizes human mesothelin, and optionally CCL19, by the immune cell. Includes. In some embodiments, the immune cell is a T cell, natural killer (NK) cell, B cell, antigen presenting cell, or granulocyte, optionally a T cell or NK cell.

pharmaceutical composition

In certain embodiments, the immune cells described above are formulated as pharmaceutical compositions. In some embodiments, the pharmaceutical composition is administered to the subject by multiple routes of administration, including, but not limited to, parenteral, oral, sublingual, or transdermal routes of administration. In some embodiments, parenteral administration is intravenous, subcutaneous, intramuscular, intranasal, intra-arterial, intra-articular, intradermal, intraosseous injection, intraperitoneal, subarachnoid, intracranial, intrasynovial, intratumoral, intradermal. , including intramedullary, intracardiac or intrathecal administration. In some embodiments, the pharmaceutical composition is formulated for topical administration. In other embodiments, the pharmaceutical composition is formulated for systemic administration.

In some embodiments, the pharmaceutical composition includes pharmaceutically acceptable excipients. Examples of additives include saline, buffered saline, cell culture medium, dextrose, water for injection, glycerol, ethanol, stabilizers, solubilizers, surfactants, buffers, preservatives, thickening agents, fillers, lubricants, or combinations thereof. can do.

In some embodiments, the pharmaceutical composition comprises acids such as acetic acid, boric acid, citric acid, lactic acid, phosphoric acid, and hydrochloric acid, bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, sodium lactate, and It further includes a pH adjuster or buffer, including Tris-hydroxymethylaminomethane, and buffers such as citrate/dextrose, sodium bicarbonate, and ammonium chloride. These acids, bases and buffers are included in the amounts necessary to maintain the pH of the composition in an acceptable range.

In some embodiments, the pharmaceutical composition includes one or more salts in the amount necessary to bring the osmolality of the composition into an acceptable range. Such salts include those having sodium, potassium or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate or bisulfite anions, suitable salts being sodium chloride, potassium chloride, Includes sodium thiosulfate, sodium bisulfite and ammonium sulfate.

In an exemplary method, the pharmaceutical composition of the present invention is administered in one portion or several divided portions, 4 times, 3 times, 2 times or 1 time per day, 1-day, 2-day, 3-day. -days, at 4-day or 5-day intervals, once a week, at 7-day, 8-day or 9-day intervals, twice a week, once a month, twice a month, once a month Can be administered three or more times independently.

If the patient's condition improves, at the discretion of the physician, administration of the composition may be given continuously or, alternatively, the dose of the composition to be administered is temporarily reduced or suspended for a certain length of time (i.e., a “dose holiday”) ). In some embodiments, the length of the drug holiday is, by way of example only, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, It can vary from 2 days to 1 year, including 50, 70, 100, 120, 150, 180, 200, 250, 280, 300, 320, 350 or 365 days. Dose reductions during the drug holiday period are just examples: 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 10%-100%, including 75%, 80%, 85%, 90%, 95% or 100%.

In some embodiments, the amount of CAR-T cells given that corresponds to this amount will vary depending on factors such as the severity of the disease and the identity (e.g., body weight) of the subject or host in need of treatment, but will nonetheless be administered, e.g. It is routinely determined in ways known in the art depending on the specific circumstances surrounding the case, including the specific agent, route of administration, and subject or host to be treated. In some embodiments, the desired dose is conveniently presented as a single dose or as divided doses administered simultaneously (or over a short period of time) or at appropriate intervals, e.g., two, three, four or more sub-doses per day. .

Because the number of variables associated with an individual treatment regimen is large and significant variations from these recommended values are not uncommon, the foregoing ranges are only indicative. Such dosages vary depending on a number of variables, including but not limited to the activity of the compound used, the disease or condition being treated, the mode of administration, the needs of the individual subject, the severity of the disease or condition being treated, and the judgment of the practitioner.

In some embodiments, the toxicity and therapeutic efficacy of such therapeutic regimens can be measured by determining the LD50 (lethal dose in 50% of the population) and ED50 (therapeutically effective dose in 50% of the population). Determined by standard pharmaceutical procedures in culture or laboratory animals. The dose ratio between toxic and therapeutic effects is the therapeutic index, which is expressed as the ratio between LD50 and ED50. Compounds showing a high therapeutic index are preferred. Data obtained from cell culture assays and animal studies are used to formulate various dosages for use in humans. The dosage of these compounds is preferably within a range of circulating concentrations that include the ED50 with minimal toxicity. Dosages will vary within this range depending on the dosage form used and the route of administration utilized.

Kits/Manufactured Supplies

In certain embodiments, a nucleic acid molecule described above, a vector comprising a nucleic acid molecule described above, a CAR that specifically recognizes mesothelin (e.g., human mesothelin), an immune cell expressing IL-15 and optionally CCL19, or a pharmaceutical Kits comprising the compositions are disclosed herein. In some embodiments, a kit may contain one or more packaging materials, such as a package insert, label, package, etc., that specify methods of use, etc. for use in the treatment of cancer. Since the immune cells in the pharmaceutical composition of the present invention have a suppressive effect on tumor recurrence, the pharmaceutical composition of the present invention can serve as a pharmaceutical composition for use in suppressing tumor recurrence. Such pharmaceutical compositions for use in suppressing tumor recurrence may contain one or more packaging materials, such as a package insert, label or package, etc., specifying the method of use for use in suppressing tumor recurrence, etc.

The term “packaging material” refers to the physical structure that houses the components of the kit. The materials can keep the components sterile and can be made of materials commonly used for this purpose (e.g. paper, corrugated fiber, glass, plastic, foil, ampoules, vials, tubes, etc.).

Kits of the present invention may include labels or inserts. A label or insert includes “print”, such as paper or cardboard, either separately or attached to a component, kit or packaging material (such as a box), or attached to an ampoule, tube or vial containing a kit component. The label or insert may additionally be a computer-readable medium, such as a disk (e.g., floppy diskette, ZIP disk), an optical disk, such as a CD- or DVD-ROM/RAM, DVD, MP3, magnetic tape, or an electrical storage medium, such as, RAM and ROM, or hybrids thereof, such as magnetic/optical storage media, FLASH media, or memory type cards.

The label or insert may contain clinical pharmacology, pharmacokinetic and pharmacodynamic identification information of the active agent(s), including one or more components therein (e.g., binding agent or pharmaceutical composition), dosage, and mechanism of action. The label or insert may include information identifying the manufacturer's information, lot number, and location and date of manufacture.

The label or insert may include information about the disease for which the kit components may be used. The label or insert may contain instructions for a clinician or subject to use one or more of the kit components in a method, treatment protocol, or therapeutic regimen. The instructions may include dosage, frequency or duration, and instructions for practicing any of the methods, treatment protocols or therapeutic regimens described herein.

The label or insert may include information about any benefits that the component may provide, such as therapeutic benefits. The label or insert may include information about potential adverse side effects, such as warning the subject or clinician about situations in which it is not appropriate to use a particular composition (e.g., a modified immune cell described herein). For example, since adverse side effects are generally more likely to occur at higher doses, frequencies, or durations of the active agent, the instructions may include recommendations for higher doses, frequencies, or durations. The subject has taken, will take, or is currently taking one or more other medications that are incompatible with the composition, or the subject has undergone, will undergo, or is currently undergoing another treatment protocol or therapeutic regimen that is incompatible with the composition. However, since adverse side effects may also occur, the instructions may include information regarding these incompatibilities.

Justice

As used in the specification and claims, the singular forms “a,” “an,” and “the” include plural referents unless the context clearly dictates otherwise. For example, the term “cell” includes a plurality of cells, including mixtures thereof.

As used herein, the term “comprising” is intended to mean that the compositions and methods include the listed elements but do not exclude others. “Consisting essentially of” when used to define compositions and methods shall mean excluding any other element of essential significance to the combination for the intended use. For example, a composition consisting essentially of elements as defined herein will not exclude trace contaminants from isolation and purification methods and pharmaceutically acceptable carriers such as phosphate buffered saline and preservatives. “Consisting of” shall mean excluding the substantial method steps for administering the compositions disclosed herein and other ingredients in excess of trace elements. The aspects defined by each of these transition terms are within the scope of this disclosure.

As used herein, the term “about” is used to indicate that a value includes the standard deviation of error for the device or method that will be used to determine the value. When used before numerical designations, including ranges, such as temperature, time, amount and concentration, the term "about" means (+) or (-) 15%, 10%, 5%, 3%, 2% or 1%. It represents an approximation that may vary.

Also, as used herein, “and/or” refers to and includes any and all possible combinations of one or more of the associated listed items as well as lack of combinations when interpreted as an alternative (“or”).

As used herein, “optionally” or “optionally” means that a subsequently described event or circumstance may or may not occur, and the description refers to embodiments in which the event or circumstance occurs and embodiments in which it does not occur. It means including.

As used herein, the term “antibody” refers to a protein that binds to another molecule (an antigen, such as mesothelin) through the heavy and light chain variable domains, V _H and V _L , respectively. The term “variable region” or “variable domain” refers to the domain of an antibody that is involved in the binding of the antibody to its antigen. The variable domains of the heavy and light chains (VH and VL, respectively) of natural antibodies generally have similar structures, with each domain containing four conserved framework regions (FR) and three CDRs. (See, e.g., Kindt et al. Kuby Immunology, 6th ed., WH Freeman and Co., page 91 (2007)). A single VH or VL domain may be sufficient to confer antigen-binding specificity. Additionally, antibodies that bind to a particular antigen can be isolated using the VH or VL domains from antibodies that bind the antigen to screen libraries of complementary VL or VH domains, respectively. See, e.g., Portolano et al., J. Immunol. 150:880-887 (1993); See Clarkson et al., Nature 352:624-628 (1991).

Antibodies of the present disclosure include monoclonal antibodies. The term “monoclonal” when used in relation to an antibody refers to an antibody that is based on, obtained from, or derived from a single clone, including any eukaryotic, prokaryotic, or phage clone. Accordingly, “monoclonal” antibodies are defined herein structurally, not the method of producing them.

Monoclonal antibodies are prepared by methods known in the art (Kohler et al., Nature , 256:495 (1975); and Harlow and Lane , Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory, 1999). Briefly, monoclonal antibodies can be obtained by injecting the antigen into mice. Polypeptides or peptides used to immunize animals may be derived from translated DNA or may be chemically synthesized and conjugated to a carrier protein. Commonly used carriers that are chemically coupled to the immunizing peptide include, for example, keyhole limpet hemocyanin (KLH), thyroglobulin, bovine serum albumin (BSA), and tetanus toxoid. Antibody production involves analyzing serum samples, removing the spleen to obtain B lymphocytes, fusing the B lymphocytes with myeloma cells to produce hybridomas, cloning the hybridomas, and producing positive clones that produce antibodies against the antigen. is selected and verified by isolating the antibody from the hybridoma culture. Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of established techniques, including, for example, affinity chromatography using Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. (e.g., Coligan et al., Current Protocols in Immunology sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; and Barnes et al. , "Methods in Molecular Biology," 10:79-104, (see Humana Press (1992)).

Antibodies of the present disclosure may belong to any antibody class, IgM, IgG, IgE, IgA, IgD or subclass. Exemplary subclasses for IgG are IgG ₁ , IgG ₂ , IgG ₃ , and IgG ₄ .

Antibodies of the present disclosure may be humanized antibodies. The term “humanized” refers to non-human amino acid residues in one or more complementarity determining regions (CDRs) that specifically bind to an antigen in a recipient human immunoglobulin molecule, and one or more human amino acids in the framework regions (FR) flanking the CDRs. refers to an antibody sequence having residues. Any mouse, rat, guinea pig, goat, non-human primate (e.g., ape, chimpanzee, macaque, orangutan, etc.) or other animal antibody can be used as a CDR donor to produce humanized antibodies. Human framework region residues can be replaced with corresponding non-human residues (e.g., from a donor variable region). Accordingly, residues in the human framework regions can be replaced with corresponding residues from a non-human CDR donor antibody. Humanized antibodies may contain residues not found in the human antibody or donor CDR or framework sequences. The use of antibody components derived from humanized monoclonal antibodies reduces problems associated with immunogenicity in non-human regions. Methods for producing humanized antibodies are known in the art (e.g., US Pat. Nos. 5,225,539; 5,530,101, 5,565,332 and 5,585,089; Riechmann et al., (1988) Nature 332:323; EP 239,400; W091/09967; EP 592,106; EP 519,596; Padlan Molecular Immunol. (1991) 28:489; Studnicka et al., Protein Engineering (1994) 7:805; Singer et al., J. Immunol. (1993) 150:2844; and Roguska et al. ., Proc. Nat'l. Acad. Sci. USA (1994) 91:969).

Antibodies of the present disclosure may be chimeric antibodies. The term “chimeric antibody” refers to an antibody in which different portions are derived from different animal species, such as those having variable regions derived from a murine monoclonal antibody and human immunoglobulin constant regions, such as humanized antibodies. In some embodiments, a technique developed for the production of “chimeric antibodies” by splicing genes from mouse antibody molecules of appropriate antigen specificity together with genes from human antibody molecules of appropriate biological activity (Morrison et al., 1984 , Proc. Natl. Acad. Sci. 81:851-855; Neuberger et al., 1984, Nature 312:604-608; Takeda et al., 1985, Nature 314:452-454) are used.

Antibodies of the present disclosure include binding fragments thereof. Exemplary antibody fragments include Fab, Fab', F(ab') ₂ , Fv, Fd, single-chain Fv (scFv), disulfide-linked Fv (sdFv), light chain variable region V _L , heavy chain variable region V _H , triple Specific (Fab ₃ ), bispecific (Fab ₂ ), diabody ((V _L -V _H ) ₂ or (V _H -V _L ) ₂ ), triabody (trivalent), tetrabody (tetravalent) , minibody ((scF _V -C _H ) ₂ ), bispecific single-chain Fv (Bis-scFv), IgGdeltaCH2, scFv-Fc, (scFv) ₂ -Fc and IgG4PE. Such fragments may have the binding affinity of a full-length antibody, the binding specificity of a full-length antibody, or one or more activities or functions of a full-length antibody, such as a function or activity of a mesothelin binding antibody.

Antibody fragments can be combined. For example, V _L or V _H subsequences can be joined by a linker sequence to form a V _L -V _H chimera. Combinations of single-chain Fvs (scFv) sequences can be joined by linker sequences to form scFv - scFv chimeras. Antibody fragments include single-chain antibodies or variable region(s) alone or in combination with all or part of other sequences.

Antibody fragments can be prepared by a variety of techniques, including, but not limited to, proteolytic digestion of intact antibodies as well as production by recombinant host cells. In some embodiments, the antibody is produced by enzymatic digestion of an intact antibody that does not occur naturally and/or contains two or more antibody regions or chains joined by a synthetic linker, such as a peptide linker. It is a recombinantly produced fragment, such as a fragment containing a sequence that may not occur. In some embodiments, the antibody fragment is an scFv.

Antibody fragments can also be prepared by proteolytic hydrolysis of the antibody, for example, pepsin or papain digestion of the whole antibody. The antibody fragment produced by enzymatic cleavage using pepsin provides the 5S fragment designated as F(ab') ₂ . This fragment can be further cleaved using a thiol reducing agent to produce the 3.5S Fab' monovalent fragment. Alternatively, enzymatic cleavage using pepsin directly produces two monovalent Fab' fragments and an Fc fragment (e.g., U.S. Pat. Nos. 4,036,945 and 4,331,647; and Edelman et al. , Methods Enymol. 1:422 (1967) reference). Other methods of antibody cleavage may also be used, such as separation of the heavy chains to form monovalent light-heavy chain fragments, further cleavage of the fragments, or other enzymes or chemicals.

In some embodiments, techniques described for the production of single chain antibodies (US Pat. No. 4,694,778; Bird, 1988, Science 242:423-42; Huston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879) -5883; and Ward et al., 1989, Nature 334:544-54) are adapted to produce single chain antibodies. Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region through amino acid bridges, resulting in a single chain polypeptide. Techniques for assembling functional Fv fragments in E. coli are also optionally used (Skerra et al., 1988, Science 242:1038-1041).

As used herein, when used in the context of two or more nucleic acid or polypeptide sequences, “identical,” “sequence identity,” or “percent identity” refers to a specified percentage of nucleotides or amino acid residues that are identical or identical over a specified region, e.g. , 60% or more identity, preferably at least 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98 Refers to two or more sequences having %, 99% or greater identity. Alignment and sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology (Ausubel et al., eds. 1987) Appendix 30, Section 7.7.18, Table 7.7.1. there is. Preferably, default parameters are used for sorting. The preferred alignment program is BLAST with default parameters. In particular, the preferred programs are BLASTN and BLASTP, which use the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; matrix = BLOSUM62; Description = 50 sequences; Sort by = high score; Database = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translation + SwissProtein + SPupdate + PIR. Detailed descriptions of these programs can be found on the Internet at: ncbi.nlm.nih.gov/cgi-bin/BLAST. The terms “identical,” “sequence identity,” or “percent identity” may also refer to or be applied to the complement of a test sequence. The term also includes sequences with deletions and/or additions as well as those with substitutions. As described herein, preferred algorithms may fill gaps, etc. Preferably, the identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is at least 50-100 amino acids or nucleotides in length. An “unrelated” or “non-homologous” sequence shares less than 40% identity, or alternatively, less than 25% identity with one of the sequences disclosed herein.

The terms “protein,” “peptide,” and “polypeptide” are used interchangeably and in the broadest sense to refer to compounds of two or more subunit amino acids, amino acid analogs, or peptidomimetics. Subunits may be linked by peptide bonds. In other embodiments, subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain two or more amino acids, and there is no limit to the maximum number of amino acids that a protein sequence or peptide sequence can contain. Polypeptides include full-length native polypeptides and “modified” forms such as subsequences, variant sequences, fusion/chimeric sequences, and dominant-negative sequences. As used herein, the term “amino acid” refers to natural and/or unnatural or synthetic amino acids, including glycine and both D and L optical isomers, amino acid analogs and peptidomimetics.

Peptides include L- and D-isomers, and combinations thereof. Peptides may include modifications typically associated with post-translational processing of proteins, such as cyclization (e.g., disulfide or amide bond), phosphorylation, glycosylation, carboxylation, ubiquitination, myristylation, or lipidation. there is. A modified peptide may have one or more amino acid residues substituted for another residue, added to the sequence, or deleted from the sequence. Specific examples include one or more amino acid substitutions, additions or deletions (e.g., 1-3, 3-5, 5-10, 10-20 or more).

As used herein, the terms “modification” and “modified” refer to a mutation in one or more amino acid residues of an antibody, protein or polypeptide compared to an equivalent reference antibody, protein or polypeptide without the modification; Refers to substitution, addition, or deletion. In some embodiments, modifications include conservative substitutions.

A “conservative substitution” is the replacement of one amino acid with a biologically, chemically, or structurally similar residue. Biologically similar means that the substitution is compatible with the activity or function of the unsubstituted sequence. Structurally similar means that the amino acids are of similar length or have side chains of similar size, such as alanine, glycine, and serine. Chemical similarity means that the residues have the same charge or are both hydrophilic or hydrophobic. Specific examples include substitution of one hydrophobic residue, such as isoleucine, valine, leucine or methionine, for another, or substitution of one polar residue for another, such as substitution of arginine for lysine, substitution of glutamic acid for aspartic acid. , or substitution of glutamine with asparagine, and substitution of serine with threonine.

As used herein, the term “nucleic acid” refers to DNA or RNA containing natural, synthetic or artificial nucleotide analogs or bases. In some embodiments, the nucleotide analog or artificial nucleotide base comprises a nucleic acid with a modification in the 2' hydroxyl group of the ribose moiety. In some embodiments, the modification includes H, OR, R, halo, SH, SR, NH ₂ , NHR, NR ₂ or CN, where R is an alkyl moiety. Exemplary alkyl moieties include, but are not limited to, halogens, sulfur, thiols, thioethers, thioesters, amines (primary, secondary, or tertiary), amides, ethers, esters, alcohols, and oxygen. In some embodiments, the alkyl moiety further comprises modifications. In some embodiments, the modification is an azo group, keto group, aldehyde group, carboxyl group, nitro group, nitroso, group, nitrile group, heterocycle (e.g., imidazole, hydrazino or hydroxylamino) group, isocyanate or cyanate groups, or sulfur containing groups (such as sulfoxides, sulfones, sulfides or disulfides). In some embodiments, the alkyl moiety further includes hetero substitution. In some embodiments, the carbon of the heterocyclic group is substituted with nitrogen, oxygen, or sulfur. In some embodiments, heterocyclic substitutions include, but are not limited to, morpholino, imidazole, and pyrrolidino.

In some embodiments, the nucleotide analog is a modified base, such as, but not limited to, 5-propynyluridine, 5-propynylcytidine, 6-methyladenine, 6-methylguanine, N,N,-dimethyladenine, 2 -propyladenine, 2-propylguanine, 2-aminoadenine, 1-methyl inosine, 3-methyluridine, 5-methylcytidine, 5-methyluridine and other nucleotides with modifications at the 5 position, 5-(2- Amino) propyluridine, 5-halocytidine, 5-halouridine, 4-acetylcytidine, 1-methyladenosine, 2-methyladenosine, 3-methylcytidine, 6-methyluridine, 2-methylguanosine , 7-methylguanosine, 2,2-dimethylguanosine, 5-methylaminoethyluridine, 5-methyloxyuridine, deazanucleotides (e.g., 7-deaza-adenosine, 6-azouridine, 6-azouridine) -azocitidine or 6-azothymidine), 5-methyl-2-thiouridine, other thio bases (e.g., 2-thiouridine, 4-thiouridine and 2-thiocitidine), dihydrouri Dean, pseudouridine, cuosine, archaeosine, naphthyl and substituted naphthyl groups, optional O- and N-alkylated purines and pyrimidines (e.g. N6-methyladenosine, 5-methylcarbonylmethyluridine , uridine 5-oxyacetic acid, pyridin-4-one or pyridin-2-one), phenyl and modified phenyl groups such as aminophenol or 2,4,6-trimethoxy benzene, which act as G-clamp nucleotides. These include modified cytosine, 8-substituted adenine and guanine, 5-substituted uracil and thymine, azapyrimidine, carboxyhydroxyalkyl nucleotides, carboxyalkylaminoalkyl nucleotides, and alkylcarbonylalkylated nucleotides. Modified nucleotides also include nucleotides that are modified with respect to the sugar moiety, as well as nucleotides having a non-ribosyl sugar or analog thereof. For example, in some embodiments the sugar moieties are or are based on mannose, arabinose, glucopyranose, galactopyranose, 4'-thioribose and other sugars, heterocycles or carbocycles. The term nucleotide also includes what are known in the art as universal bases. By way of example, and not limited to, common bases include 3-nitropyrrole, 5-nitroindole, or nebularine.

The nucleic acid molecule of the present invention can be produced by publicly known techniques such as chemical synthesis or PCR amplification based on information about the nucleotide sequence of each nucleic acid. The codon selected to encode an amino acid can be manipulated to optimize nucleic acid expression in the host cell of interest.

In the present invention, “expression of IL-15” by a T cell means that various forms of IL-15 (e.g., wild type, variants or fusion proteins described above) can be expressed, as long as the effect of the present invention is not lost. it means.

As used herein, the term "substantially" when describing a population of T cells means that it contains less than about 30%, 25%, 20%, 15%, 10%, or 5% contaminating cells. refers to a group. In some embodiments, contaminating cells are less than about 20% of the population of T cells. In some embodiments, contaminating cells are less than about 15% of the population of T cells. In some embodiments, contaminating cells are less than about 10% of the population of T cells.

As used herein, the terms “treating,” “treatment,” and the like refer to obtaining a desired pharmacological and/or physiological effect. The effect may be therapeutic in terms of improvement of symptoms of the disease, partial or complete cure of the disease, and/or abnormal effects due to the disease. In one aspect, the term “treatment” excludes prophylaxis.

As used herein, “treatment” further includes systemic improvement of symptoms and/or delay in onset of symptoms associated with pathology. Clinical and sub-clinical evidence of “cure” will vary depending on the pathology, individual and treatment. In one aspect, treatment excludes prevention.

The term “improve” refers to a detectable improvement in a subject's condition. Detectable improvement is a symptom caused by or associated with the disease, such as the occurrence, frequency, severity, progression, or persistence of one or more abnormal symptoms, disorders, diseases, pathologies, conditions, or complications caused by or associated with the disease. It includes subjective or objective reduction, reduction, inhibition, suppression, limitation or control of duration, or amelioration of the underlying cause or consequence of the disease, or reversal of the disease.

Accordingly, treatment includes reducing, reducing, inhibiting, suppressing, limiting, controlling or preventing the disease, or its associated symptoms or consequences, or the underlying cause; Reduce, reduce, inhibit, suppress, limit, control or prevent the progression or worsening of a disease, condition, symptom or consequence or underlying cause; or may result in further deterioration or development of one or more additional symptoms of the disease condition or condition. Accordingly, a successful treatment outcome may be due to one or more symptoms or underlying causes or consequences of the condition, disease or condition in the subject, e.g., caused by or associated with one or more abnormal symptoms, disorders, diseases, pathology, conditions, or diseases or conditions. Causes a “therapeutic effect” or “benefit” that reduces, reduces, inhibits, suppresses, limits, controls or prevents the occurrence, frequency, severity, progression or duration of associated complications. Accordingly, treatment methods that affect one or more root causes of a condition, disease, or symptom are considered beneficial. Stabilizing a disorder or condition is also a successful treatment outcome.

Accordingly, therapeutic benefit or improvement need not completely eliminate one, most or all of the symptoms, complications, consequences or root causes associated with the condition or disease. Therefore, a satisfactory endpoint may be defined as one or more of the following physiological, biochemical or cellular signs or disorders or diseases, over a short or long duration (hours, days, weeks, months, etc.), e.g. Incremental improvement of the subject's condition, or one or more associated abnormal symptoms or complications or consequences or underlying causes, exacerbations or symptoms of a symptom, disorder, disease, pathology, condition, or complication caused by or associated with the disease or condition When there is a partial slowing, reduction, inhibition, suppression, limitation, control or prevention of the occurrence, frequency, severity, progression or duration of a progression (e.g., stabilizing one or more symptoms or complications of a condition, disorder or disease); achieved.

When used to describe the selection of any element, range, dosage form, etc. disclosed herein, the terms "acceptable," "effective," or "sufficient" mean that the element, range, dosage form, etc. is suitable for the purpose for which it is disclosed. It means that

The terms “subject,” “host,” “individual,” and “patient” are used interchangeably herein to refer to animals, typically mammals. Any suitable mammal can be treated by the methods, cells or compositions described herein. Non-limiting examples of mammals include humans, non-human primates (e.g., apes, gibbons, chimpanzees, orangutans, monkeys, and macaques, etc.), livestock (e.g., dogs and cats), and farm animals (e.g., horses, cattle, etc.). , goats, sheep, pigs) and laboratory animals (e.g., mice, rats, rabbits, guinea pigs). In some embodiments, the mammal is a human. The mammal can be of any age or at any stage of development (eg, adult, teenage, infant, infant, or intrauterine mammal). Mammals can be male or female. Mammals may be pregnant females. In some embodiments, the subject is a human.

As used herein, the term “γδT cell” refers to a cell that expresses CD3 and a TCR consisting of a TCRγ chain (γTCR) and a TCRδ chain (δTCR) (hereinafter sometimes referred to as “γδTCR”) .

As used herein, the term “αβT cell” refers to a cell that expresses CD3 and a TCR consisting of a TCRα chain (αTCR) and a TCRβ chain (βTCR) (hereinafter sometimes referred to as “αβTCR”) . In some cases, almost all αβT cells recognize antigenic peptide-MHC (major histocompatibility complex, in humans, HLA: human leukocyte antigen) complexes by the αβTCR (this is referred to as MHC restriction). In contrast, γδT cells recognize a variety of molecules expressed by the cells by the γδTCR, regardless of the MHC molecule.

As used herein, the term “modulate” or “modulator” refers to the ability of an element to positively or negatively alter its associated function. Exemplary adjustments include changes of about 1%, about 2%, about 5%, about 10%, about 25%, about 50%, about 75%, or about 100%.

In certain embodiments, provided herein are TGFβ signaling modulators that can alter or prevent signaling of the TGFβ receptor. Those skilled in the art will understand that this can be achieved by binding to a cytokine that activates signaling of the TGFβR (i.e., TGFβ) or to the receptor itself (e.g., a TGFβ antibody or fragment thereof, a TGFBR antibody or fragment thereof). Accordingly, this term includes both molecules that bind to TGFβ and molecules that bind to TGFβR. In one embodiment, a modulator of the present disclosure is capable of neutralizing TGFβ signaling via TGFβRII. “Neutralization” means that the normal signaling effects of TGFβ are blocked such that the presence of TGFβ has a neutral effect on TGFβRII signaling. In some embodiments, the TGFβ modulator improves the host's immune response.

As used herein, the terms “transforming growth factor-β”, “TGF-beta”, “TGFb”, “TGFβ”, and “transforming growth factor-beta” are used interchangeably herein and refer to latent forms and Refers to a family of molecules with the full-length native amino acid sequence of any of the TGF-betas from humans, including precursors and associated or unassociated complexes of mature TGF-betas (“latent TGF-betas”). Reference to such TGF-beta herein refers not only to any of the currently identified forms, including TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4 and TGF-beta5 and their latent versions. derived from the sequence of any known TGF-beta and at least about 75%, preferably at least about 80%, more preferably at least about 85%, even more preferably at least about 90%, and even more preferably will be understood as a reference to the later identified human TGF-beta species comprising polypeptides that are at least about 95% homologous. The specific terms “TGF-beta1”, “TGF-beta2” and “TGF-beta3” as well as “TGF-beta4” and “TGF-beta5” are used in literature, e.g., Derynck et al., supra, Nature , Seyedin et al., J. Biol, above. Chem., 262, and TGF-beta as defined in deMartin et al., supra. The term “TGF-beta” refers to the gene encoding human TGF-beta. The preferred TGF-beta is native-sequence human TGF-beta.

Members of the TGF-beta family are defined as those that have nine cysteine residues in the mature part of the molecule, share more than 65% homology with other known TGF-beta sequences in the mature region, and are able to compete for the same receptor. . Moreover, they all appear to be encoded as larger precursors that share a region of high homology near the N-terminus and show conservation of three cysteine residues in the part of the precursor that would later be removed by processing. Moreover, TGF-beta appears to have a processing site containing 4 or 5 amino acids.

As used herein, the terms “transforming growth factor-β receptor”, “TGF-bR” or “TGF-b receptor” or “TGF-beta receptor” or “TGFβR” refer to three sub-types of the TGFβR family ( That is, TGFβR1, TGFβR2, TGFβR3) are all used to include them. TGFβ receptors are characterized by serine/threonine kinase activity and exist in several different isoforms that can be homodimers or heterodimers.

As used herein, the terms “TGFβ signaling pathway modulator,” “TGFβ modulator,” “TGFβ signaling pathway modulator,” or “TGFβ modulator,” as used interchangeably herein, refer to a substance capable of modulating the TGFβ signaling pathway ( refers to a molecule (e.g., an antibody or fragment thereof), which may bind to TGFβ itself, or to a TGFβ receptor on a cell. In either case, the modulator inhibits the TGFβ signaling pathway (e.g., by binding to the cytokine (i.e., TGFβ) itself or by binding to the receptor for TGFβ). Therefore, this term includes both types of modulators that bind to TGFβ and modulators that bind to the TGFβ receptor. In various embodiments described herein, the TGFβ signaling pathway modulator is expressed together with a chimeric antigen receptor in modified immune cells (e.g., CAR-T cells). In various embodiments described herein, the TGFβ signaling pathway modulator is secreted from transformed immune cells or is expressed bound to the membrane of the immune cell. CAR-T cells expressing these TGFβ signaling pathway modulators are referred to herein as TGFβ armed CAR-T cells.

As used herein, the term “induced pluripotent stem cell” (hereinafter sometimes referred to as “iPS cell”) is established by introducing reprogramming factors into somatic cells and is capable of differentiation into many of the cells present in a living organism. It refers to stem cells that have pluripotency and the ability to proliferate. This includes any cells derived from hematopoietic progenitor cells to be used in the present invention. The induced pluripotent stem cells are preferably derived from mammals (e.g. mice, rats, hamsters, guinea pigs, dogs, monkeys, orangutans, chimpanzees, humans), more preferably from humans.

In some cases, pluripotent stem cells can be induced or established by introducing reprogramming factors into any somatic cell. Exemplary reprogramming factors include genes and gene products such as Oct3/4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas , ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1, etc. These reprogramming factors can be used alone or in combination. In some cases, combination reprogramming factors include WO 2007/069666, WO 2008/118820, WO 2009/007852, WO 2009/032194, WO 2009/058413, WO 2009/057831, WO 2009/075119, WO 2009/ 079007, W.O. 2009/091659, WO 2009/101084, WO 2009/101407, WO 2009/102983, WO 2009/114949, WO 2009/117439, WO 2009/126250, WO 2009/126251, WO 200 9/126655, WO 2009/157593, WO 2010/009015, WO 2010/033906, WO 2010/033920, WO 2010/042800, WO 2010/050626, WO 2010/056831, WO 2010/068955, WO 2010/098419, WO 201 0/102267, WO 2010/111409, WO 2010/111422, WO 2010/115050, WO 2010/124290, WO 2010/147395, WO 2010/147612, Huangfu D, et al. (2008), Nat. Biotechnol., 26: 795-797, Shi Y, et al. (2008), Cell Stem Cell, 2: 525-528, Eminli S, et al. (2008), Stem Cells. 26:2467-2474, Huangfu D, et al. (2008), Nat. Biotechnology. 26:1269-1275, Shi Y, et al. (2008), Cell Stem Cell, 3, 568-574, Zhao Y, et al. (2008), Cell Stem Cell, 3:475-479, Marson A, (2008), Cell Stem Cell, 3, 132-135, Feng B, et al. (2009), Nat. Cell Biol. 11:197-203, R.L. Judson et al., (2009), Nat. Biotechnol., 27:459-461, Lyssiotis CA, et al. (2009), Proc Natl Acad Sci U S A. 106:8912-8917, Kim JB, et al. (2009), Nature. 461:649-643, Ichida JK, et al. (2009), Cell Stem Cell. 5:491-503, Heng JC, et al. (2010), Cell Stem Cell. 6:167-74, Han J, et al. (2010), Nature. 463:1096-100, Mali P, et al. (2010), Stem Cells. 28:713-720, and Maekawa M, et al. (2011), Nature. Illustrated by the combination described in 474:225-9.

Examples of somatic cells include, but are not limited to, any of fetal somatic cells, neonatal somatic cells, and mature somatic cells, as well as any of primary cultured cells, subcultured cells, and established cell lines. Additionally, the cells described above may be healthy cells or diseased cells. Specific examples of somatic cells include (1) tissue stem cells (somatic stem cells), such as neural stem cells, hematopoietic progenitor cells, mesenchymal stem cells, and dental pulp stem cells; (2) tissue progenitor cells; and (3) differentiated cells, such as blood cells (e.g., peripheral blood cells and cord blood cells, etc.), monocytes (e.g., lymphocytes (NK cells, B cells, T cells other than αβT cells (e.g., γδT cells, etc.)) , monocytes, and dendritic cells, etc.), granulocytes (e.g., eosinophils, neutrophils, basophils), megakaryocytes), epithelial cells, endothelial cells, muscle cells, fibroblasts (e.g., skin cells, etc.), hair cells, liver cells, Includes gastric mucosal cells, enterocytes, spleen cells, pancreatic cells (e.g., pancreatic exocrine cells, etc.), brain cells, lung cells, kidney cells, and adipocytes. Among these, monocytes other than αβT cells are preferable, and more specifically, monocytes or γδT cells are preferable.

Exemplary methods, such as calcium phosphate coprecipitation, PEG methods, electroporation, microinjection, and lipotransfection, can be used to introduce reprogramming factors into somatic cells. Additional methods can be found in Cell Engineering additional volume 8, New Cell Engineering experiment protocol, 263-267 (1995) (published by Shujunsha), Virology, vol. 52, 456 (1973), and Folia Pharmacol. Jpn., vol. 119 (No. 6), 345-351 (2002). If viral vectors are used, introduce the nucleic acids into suitable packaging cells (e.g., Plat-E cells) and complementing cell lines (e.g., 293 cells), recover the viral vectors produced in the culture supernatant, and introduce the respective viral vectors into the cells. Infect the vector using an appropriate method to introduce the vector into the cell. For example, when a retroviral vector is used as a vector, specific means are disclosed in WO 2007/69666, Cell, 126, 663-676 (2006) and Cell, 131, 861-872 (2007). In particular, when retroviral vectors are used, highly efficient transfection into a variety of cells is possible by using the recombinant fibronectin fragment CH-296 (manufactured by Takara Bio Inc.).

Reprogramming factors in the form of RNA can be introduced directly into cells and expressed in the cells. Known methods for introducing RNA can be used, for example, lipofection or electroporation can be preferably used. If the reprogramming factor is in the form of a protein, it can be introduced into cells by methods such as lipofection, fusion with cellular membrane-penetrating peptides (e.g., HIV-derived TAT and polyarginine), and microinjection.

In some embodiments, the method of differentiating induced pluripotent stem cells into T cells is not particularly limited, as long as the induced pluripotent stem cells can be differentiated into γδT cells. In some cases, the methods include differentiating induced pluripotent stem cells into hematopoietic progenitor cells and differentiating the hematopoietic progenitor cells into CD3 positive T cells. Exemplary methods for use in differentiating iPSCs into hematopoietic progenitor cells include WO 2013/075222, WO 2016/076415 and Liu S. et al., Cytotherapy, 17 (2015); Including those disclosed at 344-358. Exemplary methods for use in differentiating hematopoietic progenitor cells into CD3 positive T cells include those disclosed in WO 2016/076415, and WO 2017/221975. Also refer to the method disclosed in WO2020/013315.

Example

These examples are provided for illustrative purposes only and do not limit the scope of the claims provided herein.

Example 1

Method for producing cells expressing γδTCR

iPS cells provided by Kyoto University, iPS Cell Research and Application Center (Ff-I01s04 cell line: healthy Suspended cell populations differentiated from an individual's peripheral blood mononuclear cells (derived from an individual's peripheral blood mononuclear cells) were used to generate cell populations containing hematopoietic progenitor cells (HPCs). For example, the Ff-I01s04 cell line was seeded at ⁶ ml VEGF, 2 μM SB431542 was added to EB medium (10 μg/ml human insulin, 5.5 μg/ml human transferrin, 5 ng/ml sodium selenite, 2 mM L-glutamine, 45 mM α-monothioglycerol, and 50 μg/ml StemPro34) supplemented with ascorbic acid 2-phosphate, and the cells were cultured for 5 days under hypoxic conditions (5% O ₂ ) (day 5). Then, 50 ng/ml SCF, 30 ng/ml TPO, 10 ng/ml FLT-3L were added and cells were cultured for 5 - 9 days (day - 14) to yield a suspended cell population. Medium was changed every 2 or 3 days during the culture period. The resulting suspended cell population containing HPC was stained using the antibody set as shown in Table 2.

Table 2

Cell populations that underwent staining mentioned above were sorted by FACSAria. The obtained cell fraction was differentiated into lymphoid cells according to known methods (e.g., methods described in Journal of Leukocyte Biology 96(2016)1165-1175 and WO 2017/221975). For example, hematopoietic progenitor cell populations were seeded at 2000 cells/well in 48-well-plates coated with recombinant h-DLL4/Fc chimera (Sino Biological) and retronectin (Takara Bio Inc.) and incubated in 5% CO ₂ , cultured under conditions of 37°C. Medium was changed every 2 or 3 days during the culture period. αMEM medium contains 15% FBS, 2 mM L-glutamine, 100 U/ml penicillin, 100 ng/ml streptomycin, 55 μμ 2-mercaptoethanol, 50 μg/ml ascorbic acid 2-phosphate, and 10 μg/ml human insulin. , 5.5 μg/ml human transferrin, 5 ng/ml sodium selenite, 50 ng/ml SCF, 50 ng/ml IL-7, 50 ng/ml FLT-3L, 100 ng/ml TPO, 15 μM SB203580, and 30 μg/ml Contained ng/ml SDF-1α. Cells were passaged into similarly coated 48-well plates on days 7 and 14 from the start of culture. All cells were collected at day 21 from the start of culture (day 35), and the presence of CD45(+), CD3(+) fractions was confirmed by flow cytometry (BD FACSAria ^TM Fusion, manufactured by BD Biosciences). The obtained cells were seeded in a 48-well plate and cultured under conditions of 5% CO ₂ and 37°C. αMEM medium contains 15% FBS, 2 mμ L-glutamine, 100 U/ml penicillin, 100 ng/ml streptomycin, 50 μg/ml ascorbic acid 2-phosphate, 10 μg/ml human insulin, 5.5 μg/ml human transferrin, Contained 5 ng/mL sodium selenite, 500 ng/mL anti-CD3 antibody (OKT3), and 10 ng/mL IL-7. Day 27 from the start of culture all cells were collected (day 41), cells were counted by hemocytometer and stained using antibody sets as shown in Table 3.

Table 3

As a result of the staining, it was shown that cells expressing γδTCR (γδTCR positive cells) could be prepared from hematopoietic precursor cells derived from iPS cells (Ff-I01s04 cell line) (Figure 2).

Furthermore, γδTCR-positive cells contained Vδ1-positive γδT cells and Vδ2-positive γδT cells, showing that Vδ1 type and Vδ2 type γδT cells could be produced (Figure 3). (Note that iPS cell-derived γδTCR positive cells lacking KI of the γδTCR gene are also referred to herein as “iγδT cells”)

Example 2

Expansion culture and functional assessment of iγδT cells

Production of iγδT cells was performed using the same method as in Example 1 except that UCHT1 (manufactured by GeneTex) was used as the anti-CD3 antibody, and γδT derived from iPS cells (Ff-I01s04 cell line) Cells (iγδT cells) were produced.

The subsequent iγδT cells obtained were suspended at 2,000,000 cells/mL in α-MEM medium containing 15% FBS and additives containing cytokines as shown in Table 4, anti-CD3 antibody (UCHT1) and retro They were seeded on a solid-phased plate with nectin and cultured at 5% CO ₂ /37°C for 3 days. On the third day of culture, cells were collected from the plates, the number of cells was counted using NucleoCounter® NC-200 (ChemoMetec), and cells were incubated with 15% FBS and cytokines as shown in Table 5. An appropriate amount was suspended in α-MEM medium containing additives, added to a non-immobilized G-Rex (registered trademark) 6-well plate (WILSONWOLF), and cultured at 5% CO ₂ /37°C. Collect aliquots of cells from the plate 4-6 times on days 5, 6, 7, 8, 9, 10, 11, 14, and 17, and count the number of cells using a hemocytometer. did.

Anti-CD3 antibody and retronectin were immobilized on the culture plate by the following method. Anti-CD3 antibody (UCHT1 final concentration 3000 ng/mL) and retronectin (final concentration 150 μg/mL) dissolved in PBS at the required concentration were added to the plate and left at 4°C overnight. After washing with PBS, the plates were tested.

Table 4

Table 5

Proliferation of iγδT cells was observed by stimulation with anti-CD3 antibody (UCHT1) and anti-CD30 antibody (Figure 4).

Example 3

Production of iPS cell-derived Vγ9Vδ2T cells

1. Preparation of iPS cells

iPS cells were prepared similarly as described in Example 1. In particular, the Ff-I01s04 cell line provided by the iPS Cell Research and Application Center (CiRA), Kyoto University was used. iPS cells were cultured according to the “feeder-free culture of human iPS cells” protocol distributed by CiRA.

2. Differentiation of iPS cells into HPCs

Differentiation of iPS cells into hematopoietic progenitor cells (HPC) was performed according to the method disclosed in WO 2017/221975.

3. Vγ9Vδ2 gene

G115γδT cell clone-derived Vγ9Vδ2 T cell receptor (Vγ9Vδ2TCR G115) was used. The nucleic acid comprising the gene encoding Vγ9Vδ2TCR G115 encoding SEQ ID NO:44 contains the gene as shown in Table 6. Nucleic acids were artificially synthesized.

Table 6

(SEQ ID NO: 44).

4. Production of retroviral vector carrying Vγ9Vδ2 gene

The lentiviral vector pLVSIN-Ub was used, and the vector was generated by removing the sequence encoding the neomycin resistance gene from the pLVSIN-CMV Neo vector (Clontech Laboratories, Inc.) and replacing the CMV promoter with the human ubiquitin promoter. Artificial oligo DNA was synthesized according to Table 6. The Vγ9Vδ2 gene was integrated into the multi-cloning site of the pLVSIN-Ub retroviral vector. Using this plasmid and the Lenti-X ^™ 293T cell line and Lenti-X ^™ Packaging Single Shot (VSV-G) from Clontech Laboratories, Inc., the lentiviral vector pLVSIN-Ub was produced.

5. Production of iPS cell-derived Vγ9Vδ2T cells

The iPS cells prepared in 1. Preparation of iPS cells were infected with the retroviral vector carrying the Vγ9Vδ2 gene prepared in 4. Production of a retroviral vector carrying the Vγ9Vδ2 gene . These cells were differentiated into T cells according to a known method (as disclosed in WO2017/221975) in the same manner as in Example 1, to prepare iPS cell-derived Vγ9Vδ2T cells. 500 ng/mL OKT3 (TakaraBio) was used as anti-CD3 antibody in the differentiation step. (Note that iPS cell-derived Vγ9Vδ2T cells prepared from iPS cells are also referred to herein as “iγ9δ2T cells.”) The obtained iγ9δ2T cells were analyzed using a flow cytometer (BD FACSAria ^TM Fusion, manufactured by BD Biosciences). The expression of CD3, γδTCR, Vγ9, and Vδ2 on the cell membrane surface was measured (Figure 5).

Example 4

Production of iPS cell-derived anti-mesothelin-CAR/IL-15γδT cells

1. Anti-mesothelin-CAR gene

A nucleic acid containing the gene encoding anti-mesothelin-CAR was artificially synthesized. Eleven patterns of artificial nucleic acids were synthesized for transfection into iγδT cells or iγ9δ2T cells using the genes listed in Table 7.

Table 7

2. IL-15 gene

The artificial nucleic acid included genes encoding each version of IL-15. The IL-15 version of the gene is shown in Table 8.

Table 8

The first pattern of artificial nucleic acids includes, in order from the N-terminus, ssVH-P4-CD8hinge/TM-41BB-CD3z-P2A-IL2sp-mIL15/Ra.

The second pattern of artificial nucleic acids includes, in order from the N-terminus, ssVH-P4-CD8hinge/TM-41BB-CD3z-P2A-mIL15/Ra-LSP.

The third pattern of artificial nucleic acid includes, in order from the N-terminus, ssVH-P4-CD8hinge/TM-41BB-CD3z-P2A-IL15Rasp-sushi15.

The fourth pattern of artificial nucleic acid includes, in order from the N-terminus, ssVH-P4-CD8hinge/TM-41BB-CD3z-P2A-sIL15-LSP.

The fifth pattern of artificial nucleic acids includes, in order from the N-terminus, ssVH-P4-CD8hinge/TM-41BB-CD3z-P2A-IL2sp-mIL15/Ra-P2A-endosp-CCL19.

The sixth pattern of artificial nucleic acids includes, in order from the N-terminus, ssVH-P4-CD8hinge/TM-41BB-CD3z-P2A-mIL15/Ra-LSP-P2A-endosp-CCL19.

The seventh pattern of artificial nucleic acid includes, in order from the N-terminus, ssVH-P4-CD8hinge/TM-41BB-CD3z-P2A-IL15Rasp-sushi15-P2A-endosp-CCL19.

The eighth pattern of artificial nucleic acid includes, in order from the N-terminus, ssVH-P4-CD8hinge/TM-41BB-CD3z-P2A-sIL15-LSP-P2A-endosp-CCL19.

The ninth pattern of artificial nucleic acids includes, in order from the N-terminus, ssVH-P4-CD8hinge/TM-CD28-CD3z-P2A-IL15sp-sushi15-P2A-endosp-CCL19.

Additionally, two more patterns were prepared using DAP10 and TNFR2 as the intracellular domains between CD8 hinge/TM and CD3z, respectively.

The tenth pattern of artificial nucleic acid includes, in order from the N-terminus, ssVH-P4-CD8hinge/TM-DAP10-CD3z-P2A-IL2sp-mIL15/Ra.

The eleventh pattern of artificial nucleic acid includes, in order from the N-terminus, ssVH-P4-CD8hinge/TM-TNFR2-CD3z-P2A-IL2sp-mIL15/Ra.

2. Production of retroviral vector carrying anti-mesothelin-CAR gene

Phoenix A cells, a retroviral packaging cell line (ATCC), were grown to 50-70% confluency in DMEM 20% FBS and Pen/Strep. Prepare DNA complexes using the respective plasmids containing the pattern of nucleic acids containing the genes encoding the CAR constructs described above, the helper plasmids gag-pol and pVSVG, and the transduction reagent Fugene HD (Promega) according to the manufacturer's protocol. did. Viruses were harvested 16-24 hours after transfection, aliquoted and frozen for further use.

3. Production of iPS cell-derived anti-mesothelin-CAR/IL-15γδT cells

iγδT cells or iγ9δ2T cells were thawed and rested in medium (DMEM 10% FCS and additives listed in Table 10) for 3 days. Flasks were coated with retronectin (Takara) and CD3 antibody (UCHT1) overnight at 4'C. iγδT cells or iγ9δ2T cells were washed, resuspended in DMEM 10% FCS and supplements specified in Table 9, and transferred to retronectin and anti-CD3 antibody coated flasks for 72 hours for activation.

iγδT cells or iγ9δ2T cells were transduced with each pattern of nucleic acids containing genes encoding the CAR constructs described above using spinocculation. Briefly, iγδT cells or iγ9δ2T cells were transferred to retronectin-coated plates (prepared according to the manufacturer's instructions) and spinoculated (2,000 g; 32'C) with the CAR-encoding pVSVG pseudotyped virus described above. Cells were incubated overnight at 37'C 5% CO2 in DMEM 10% FCS and additives listed in Table 10, transduction was repeated the next day and cells were expanded in DMEM 10% FCS and additives listed in Table 10.

Transduced cells were further expanded using retronectin/anti-CD3 antibody activation as described above in DMEM 10% FCS and additives listed in Table 10 and transferred to G-REX at day 3 after activation. At day 7 after activation, cells were harvested, frozen, and used further. (iPS cell-derived anti-mesothelin-CAR/IL-15γδT cells prepared from iγδT cells are also referred to herein as “iMeso-CAR/IL-15γδT cells”, and iPS cell-derived Note that anti-Meso-CAR/IL-15γδT cells are also referred to herein as “iMeso-CAR/IL-15γ9δ2T cells”).

Example 5

Expansion culture of iMeso-CAR/IL-15γ9δ2T cells

Expansion culture of iMeso-CAR/IL-15γ9δ2T cells (P4-41BB-mIL15/Ra-CCL19) obtained in Example 5 was performed using methods similar to those described in Example 3. Media containing the additive containing the cytokines of Table 9 instead of the additive containing the cytokines of Table 4, and the additive containing the cytokines of Table 10 instead of the additive containing the cytokines of Table 5 were used.

Table 9

Table 10

Expansion culture of iMeso-CAR/IL-15γ9δ2T cells obtained in Example 4 was performed.

Proliferation of iMeso-CAR/IL-15γ9δ2T cells was observed by stimulation with anti-CD3 antibody (OKT3) (FIG. 6).

Example 6

1. Mesothelin expression by human cell lines MiaPaCa-2, MiaPaCa-2 Msln+, Capan-2 and GSU.

Human cell lines MiaPaCa-2 (ATCC; CRL-1420), MiaPaCa-2 Msln+ (generated from MiaPaca-2 by overexpression of mesothelin using viral vectors), Capan-2 (ATCC, HTB-80) and GSU (RIKEN , RBC2278) were cultured as recommended by the supplier. For flow cytometry, cells were harvested using TrypLE expressing enzyme (Gibco; #12605010), washed, and incubated with primary unlabeled anti-mesothelin antibody (Rockland; #MN1200301A88) and secondary APC conjugated anti-mouse IgG. Stained with antibody (Biolegend; #405308). Killed cells were stained using fixable viability dye (eBiosciences; #65-0866-14), and cells were fixed prior to acquisition on a FACS Fortessa (BD Biosciences). Analysis was performed using FlowJo. MiaPaca-2 was very low or negative for mesothelin expression, whereas Capan-2, GSU, and MiaPaca-2 Msln+ showed high mesothelin expression (Figure 7A).

2. In vitro cytotoxicity of iPS cell-derived anti-mesothelin-CAR/IL-15γδT cells against human cell lines MiaPaCa-2, MiaPaCa-2 Msln+, Capan-2 and GSU.

All target cell lines (MiaPaCa-2, MiaPaCa-2 Msln+, Capan-2, and GSU) were isolated, washed, resuspended in assay medium, counted, and plated on collagen-coated flat bottom plates in triplicate ( in triplicates) was plated. Cells were incubated at 37'C 5% CO2 for at least 2 hours to achieve complete adhesion.

Meso iCAR-Ts were thawed, washed, resuspended in assay medium, counted, and then adjusted to the indicated effector-to-target (E:T) ratio (i.e., 3:1, 1:1, 0.3:1, 0.1:1). ) and co-cultured with target cells at 37'C 5% CO2 for 48 hours. Killing of target cells by iMeso-CAR/IL-15γ9δ2T cells was assessed using the CellTiter-Glo luminescent cell viability assay (Promega) according to the manufacturer's instructions. Percent kill was calculated according to the following equation:

% killing = (average luminescent co-culture - average luminescent effector cells alone)

Average luminescence target cell alone

Results showed that iMeso-CAR/IL-15γ9δ2T cells carrying all IL-15 versions were either a cell line that naturally expressed mesothelin (GSU, Capan-2) or a cell line engineered to express mesothelin in vitro (MiaPaCa-2 Msln+). potentially killing but not mesothelin negative MiaPaCa-2 cells, showing that killing is significantly increased compared to untransduced control cells (UTD) (iγ9δ2T cells) (Figure 7b).

Example 7

1. Capan-2 s.c. Evaluating the in vivo anti-tumor efficacy of iPS cell-derived anti-mesothelin-CAR/IL-15γδT cells carrying different costimulatory domains in a xenograft model

6-16 week old female NSG mice (Jackson Labs) were inoculated subcutaneously with 4x10 ⁶ viable Capan-2 tumor cells in 100 ul McCoys 5A + matrigel. Three weeks after implantation, tumor size reached approximately 50 mm3 and mice were randomized into treatment groups with similar average tumor sizes (mean ~100 mm3; n=6 per group). The next day, 1x10 ⁶ freshly produced iMeso-CAR/IL-15γ9δ2T cells were injected intratumorally. Body weight was measured twice weekly to monitor toxicity. Tumor size was measured twice weekly, and tumor volume was calculated using the following formula: tumor volume (mm ³ ) = length x width x height x 0.5236. See Table 11 below.

Table 11

The highest anti-tumor efficacy was observed using iMeso-CAR/IL-15γδT cells carrying 4-1BB or CD28 costimulatory domains (Figure 8).

Example 8

1. Capan-2 s.c. Comparison of 4-1BB and CD28 costimulatory domains in a xenograft model and evaluating the in vivo anti-tumor efficacy of iPS cell-derived anti-mesothelin-CAR/IL-15γδT cells carrying different versions of IL-15

6-16 week old female NSG mice (Jackson Labs; n=6/group) were inoculated subcutaneously with 4x10 ⁶ viable Capan-2 tumor cells in 100 ul McCoys 5A + matrigel. When tumor size reached approximately 100 mm3 after 3 weeks of implantation, mice were randomized into treatment groups with similar average tumor size (mean -100 mm3; n=6 per group). The next day, 5x10 ⁶ (arm 1) or 1.5x10 ⁶ (arm 2) cryopreserved iMeso-CAR/IL-15γ9δ2T cells were injected intratumorally. Body weight was measured twice weekly to monitor toxicity. Tumor size was measured twice weekly, and tumor volume was calculated using the following formula: tumor volume (mm ³ ) = length x width x height x 0.5236. See Table 12-14.

Table 12: Arm 1. See also Figures 9A and 9B.

Table 13: Arm 2. See also Figures 9C and 9D.

Table 14: See Figure 9e

iMeso-CAR/IL-15γ9δ2T cells carrying IL-15 versions Sushi15 or mIL15/Ra had the highest overall anti-tumor efficacy regardless of the number of cells used (i.e., ^5x10 or ^1.5x10 ; Figure 9e). showed. iMeso-CAR/IL-15γ9δ2T cells carrying the costimulatory domain 4-1BB showed superior tumor control compared to iMeso-CAR/IL-15γ9δ2T cells using the CD28 costimulatory domain (Figure 9E).

Example 9

1. GSU s.c. Evaluating the in vivo anti-tumor efficacy of iPS cell-derived anti-mesothelin-CAR/IL-15γ9δ2T cells armored with different IL-15 versions and CCL19 in a xenograft model

6-16 week old female NSG mice (Jackson Labs; n=6/group) were inoculated subcutaneously with 2×10 ⁶ viable GSU tumor cells in Matrigel:PBS 1:1. Seven days after implantation, tumor size reached approximately 100 mm3 and mice were randomized into treatment groups with similar average tumor sizes (mean -100 mm3; n=6 per group). The next day, ^10x106 cryopreserved iMeso-CAR/IL-15γ9δ2T cells were injected intratumorally. Body weight was measured twice weekly to monitor toxicity. Tumor size was measured twice weekly, and tumor volume was calculated using the following formula: tumor volume (mm ³ ) = length x width x height x 0.5236. See Table 15 and Figure 10 below.

Table 15

Intraperitoneal administration of 10x10 ⁶ iMeso-CAR/IL-15γ9δ2T cells cleared the tumor, whereas intravenous administration of 10x10 ⁶ Meso iCAR-T resulted in tumor control but no complete response. iMeso-CAR/IL-15γ9δ2T cells armed with sushi15-CCL19 or mIL15/Ra-CCL19 performed relatively well.

Example 10

1. GSU i.p. Evaluating the in vivo anti-tumor efficacy of iPS cell-derived anti-mesothelin-CAR/IL-15γ9δ2T cells armed with different IL-15 versions and CCL19 in a xenograft model

Luciferated GSU cell lines were generated using RediFect Red-FLuc-puromycin virus (CLS960002) from Perkin Elmer. Transduction was performed overnight in the presence of 1.6 ug/ml polybrene (Millipore), and cells were transduced with virus carrying a plasmid encoding human CD19 under the control of the EF1a promoter and puromycin resistance. GSU-RedLuc cells were positively selected using puromycin.

6-16 week old female NSG (Jackson Labs; n=4/group) were inoculated intraperitoneally with 5×10 ⁶ viable GSU-RedLuc cells in Matrigel:PBS 1:1. Two days after implantation, tumor growth was monitored by bioluminescence imaging using the In Vivo Imaging System (IVIS) Spectrum (Perkin Elmer) and analyzed using IVIS Imaging software (Perkin Elmer) for randomization. Values are expressed as photons/second. Briefly, 6 min before imaging, animals received an intraperitoneal (IP) injection of 150 mg/kg D-luciferin potassium salt (Promega). Anesthesia was provided using inhaled isoflurane. The day after randomization, mice were administered 10x10 ⁶ viable iMeso-CAR/IL-15γ9δ2T cells intraperitoneally or intravenously, and tumor growth and body weight were monitored twice per week until the end of the study. See Table 16 and Figure 11 below.

Table 16

^10x106 iMeso-CAR/IL-15γ9δ2T cells strongly inhibited tumor growth in intraperitoneal GSU-RedLuc, regardless of the IL-15 version used (sushi15 vs. mIL15/Ra). iMeso-CAR/IL-15γ9δ2T cells administered intraperitoneally resulted in rapid and complete tumor elimination, whereas iMeso-CAR/IL-15γ9δ2T cells injected intravenously significantly inhibited tumor growth compared to the PBS-treated control group.

Example 11

Repeated antigen stimulation in mesothelin (Msln)-positive tumors.

Approximately 100,000 iPSC-derived anti-Msln CAR-T cells co-expressing CAR against Msln with a TGFβ modulator (e.g., TGFβR2-VH or dnTGFbR2) or control VH against GFP (Msln-control VH) Co-cultured in duplicates with 40,000 MiaPaca-2 tumor cells overexpressing human Msln in the presence or absence of TGF-β (R&D Systems, 10 ng/ml). TGFβR2-VH was secreted from CAR-T cells, whereas dnTGFbR2 was bound to the membrane of CAR-T cells. Every 3–4 days, half of the CAR-T cells per well were transferred to a new plate of tumor cells under the same conditions (with or without 10 ng/ml of TGF-β). Supernatants were harvested and frozen for later evaluation. CAR-T cells were counted by flow cytometry, and FACS phenotyping was performed at selected time points (Figure 12a).

The viability of tumor cells was assessed using CellTiterGlo (Promega) according to the manufacturer's protocol. Plates were analyzed using a Pherastar plate reader. Percent kill was estimated using the following formula:

Control wells contained only tumor cells without effector (i.e. CAR-T) cells. Percent cytotoxicity is shown in Figure 12B.

Cell counts were performed by excluding apoptotic cells using Sytox Red dye (Thermofisher, according to the manufacturer's protocol), and equal volumes of cell suspension were acquired on a Fortessa flow cytometer (BD Biosciences) using an HTS unit. Live CAR-T cells were counted by gating on live cells, single cells, and size. The results were extrapolated to obtain the number of cells per well.

After multiple rounds of restimulation with target cells simulating chronic antigen activation, TGF-β was observed to induce inhibition of CAR-T cell function (i.e. killing) and inhibit proliferation of CAR-T cells. Only CAR-T cells expressing TGFβ modulators (e.g., secreting TGFβR2 VH dimers or expressing membrane-bound dnTGFbR2) but not control VH were protected from the inhibitory effects of TGF-β (10 ng/ml).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by a person skilled in the art to which this technology pertains.

The technology illustratively described herein can suitably be practiced in the absence of any element or elements, limitation or limitations not specifically disclosed herein. Accordingly, for example, the terms “comprising,” “including,” “including,” and the like are to be read without limitation and broadly. Additionally, the terms and expressions used herein are to be used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions to exclude any equivalents or portions of the features shown and described, but rather the claimed subject matter. It is recognized that various variations are possible within the category.

Accordingly, it is to be understood that the materials, methods, and examples provided herein are representative of preferred embodiments, are illustrative, and are not intended to limit the scope of the technology.

The technology has been described broadly and generally herein. Each of the narrower species and sub-generic groupings that fall within the general disclosure also form part of the present description. This includes a general description of the subject matter along with any provisos or negative restrictions that remove any subject matter from the genus, regardless of whether the removed material is specifically listed herein.

Moreover, where features or aspects of the technology are described in terms of a Markush group, those skilled in the art will recognize that the technology is also described in terms of any individual member or subgroup of members of the Markush group.

All publications, patent applications, patents and other references mentioned herein are expressly incorporated by reference in their entirety to the same extent as if each were individually incorporated by reference. In case of conflict, the present specification, including definitions, will control.

Other embodiments are set forth within the following claims.

sequence list

summary table

SEQ ID NO: 1 / AA / P4 scFv (with/without signal peptide)

SEQ ID NO: 2 / AA / ssVH signal peptide (sp)

SEQ ID NO: 3 / AA / ssVHsp-P4 scFv

SEQ ID NO: 4 / AA / Linker (P4 scFv-CD8 hinge))

SEQ ID NO: 5 / AA / P2A peptide

SEQ ID NO: 6 / AA / IL2 sp

SEQ ID NO: 7 / AA / IL15Rasp

SEQ ID NO: 8 / AA / mIL15/Ra (including 26aa linker) (with/without IL2sp)

SEQ ID NO: 9 / AA / mIL15/Ra-LSP (IL15LSP-(IL15 propeptide)-IL15-26aa linker-IL15Ra(FL))

SEQ ID NO: 10 / AA / sushi15 (including 26aa linker) (with/without IL15Rasp)

SEQ ID NO: 11 / AA / sIL15-LSP (IL15LSP-(IL15 propeptide)-IL15)

SEQ ID NO: 47 / AA / IL15LSP

SEQ ID NO: 48 / AA / IL15 propeptide

SEQ ID NO: 12 (endosp)

SEQ ID NO: 13 / AA / CCL19

SEQ ID NO: 14 / AA / IL2sp-mIL15/Ra-P2A-endosp-CCL19 (including 26aa linker)

SEQ ID NO: 24 / AA / 4-1BB costimulatory domain

SEQ ID NO: 25 / AA / CD3z (wt) intracellular region

SEQ ID NO: 26 / AA / CD8 hinge

SEQ ID NO: 27 / AA / CD8TM domain

SEQ ID NO: 43 / AA / ssVHsp-CD8 hinge-TM-41BB-CD3z

SEQ ID NO: 42 / NA / ssVHsp-P4-CD8 Hinge-TM-BB-CD3z

SEQ ID NO: 28 / AA / IL2sp-mIL15/Ra (IL2sp-IL15-26 aa linker-IL15Ra(FL))

SEQ ID NO: 29 / AA / IL15Rasp-sushi15 (IL15Rasp-sushi domain-26 aa linker- IL15)

Peptide linker in SEQ ID NO: 45/AA/P4 scFv (20 AA)

SEQ ID NO: 31 / AA / IL15Rasp-sushi15-P2A-endosp-CCL19 (IL15Rasp-sushi domain-26 aa linker-IL15-P2A-endosp-CCL19)

SEQ ID NO: 32 / AA / peptide linker (26 AA)

SEQ ID NO: 33 / AA / peptide linker (20 AA)

SEQ ID NO: 34 / AA / ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL2sp-mIL15/Ra (including 26aa linker)

SEQ ID NO: 35 / AA / ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15Rasp-sushi15-IL15 (including 26aa linker)

SEQ ID NO: 36 / AA / ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL2sp-mIL15/Ra-P2A-endosp-CCL19 (including 26aa linker)

SEQ ID NO: 15 / AA / ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15Rasp-sushi15-IL15-endosp-CCL19 (including 26aa linker)

SEQ ID NO: 37 DNA P4-BB-mIL15/Ra

P4-BB-mIL15/Ra-LSP

SEQ ID NO: 38 DNA P4-BB-sushi15

P4-BB-sIL15-LSP

SEQ ID NO: 39 DNA P4-BB-mIL15/RaxCCL19

P4-BB-mIL15/Ra-LSPxCCL19

SEQ ID NO: 40 DNA P4-BB-sushi15xCCL19

SEQ ID NO: 50 / DNA /

>Seq42A_from_37

SEQ ID NO: 51 / DNA /

>Seq42B_from_38 (1 base difference from 42ª)

SEQ ID NO: 52 / DNA /

>Seq42C_from_39 (identity to Seq42A 1154/1523 (75.8%); identical to DNA sequence from CAR Seq ID#40)

SEQ ID NO: 44 / AA / Vg9-P2A-Vd2 TCR

SEQ ID NO: 53 AA SSVHsp-P4-BB-mIL15/Ra-LSP

(ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15propeptide-mIL15/Ra (with 26aa linker)

SEQ ID NO: 54 AA SSVHsp-P4-BB-sIL15-LSP

(ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15 Propeptide-IL15)

SEQ ID NO: 55 AA SSVHsp-P4-BB-mIL15/Ra-LSP-CCL19

(ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15 Propeptide-IL15-P2A-endospCCL19)

SEQ ID NO: 56 AA SSVHsp-P4-BB-sIL15-LSP-CCL19

(ssVHsp-P4-CD8h/TM-BB-CD3z-P2A-IL15LSP-IL15 Propeptide-IL15-P2A-endospCCL19)

SEQ ID NO: 57 AA SSVHsp-P4-CD28-sushi15-CCL19

(ssVH-P4-CD28hinge-TM-CD28cyto-CD3z-P2A-IL15Rasp-IL15Ra(sushi)-20aa linker-IL15-P2A-endospCCL19)

SEQ ID NO: 58 DNA ssVHsp-P4-BB-mIL15/Ra-LSP

SEQ ID NO: 59 DNA ssVHsp-P4-BB-sIL15-LSP

SEQ ID NO: 60 DNA ssVHsp-P4-BB-mIL15/Ra-LSP-CCL19

SEQ ID NO: 61 DNA ssVHsp-P4-BB-sIL15-LSP-CCL19

SEQ ID NO: 63 / AA / CD8TM domain

SEQ ID NO: 64 / AA / CD8 hinge

SEQ ID NO: 65 / AA / ssVHsp-P4-CD8 Hinge-TM-41BB-CD3z

SEQ ID NO: 66 / AA / ssVHsp-P4-CD8 Hinge-TM-41BB-CD3z

SEQ ID NO: 67 / AA / ssVHsp P4-CD8 Hinge-TM-41BB-CD3z

SEQ ID NO: 68 / AA / ssVHsp-P4-CD8 Hinge-TM-41BB-CD3z

SEQ ID NO: 69 / AA / ssVHsp-P4-CD8 Hinge-TM-41BB-CD3z

SEQ ID NO: 70 / AA / ssVHsp-P4-CD8 Hinge-TM-41BB-CD3z

SEQ ID NO: 71 / AA / ssVHsp-P4-CD8 hinge-TM-41BB-CD3z

SEQ ID NO: 72 / AA / ssVHsp-P4-CD8 Hinge-TM-41BB-CD3z

SEQ ID NO: 73 / AA / ssVHsp-P4-CD8 hinge-TM-41BB-CD3z

SEQ ID NO: 74 / AA / ssVHsp-P4-CD8 Hinge-TM-41BB-CD3z

SEQ ID NO: 75 / AA / ssVHsp-P4-CD8 Hinge-TM-41BB-CD3z

Claims (67)

A first polynucleotide encoding a chimeric antigen receptor (CAR) comprising an antibody that specifically recognizes human mesothelin and a 4-1BB intracellular region, and a second polynucleotide encoding interleukin 15 (IL-15) An isolated nucleic acid molecule, comprising: According to paragraph 1,
An isolated nucleic acid molecule, wherein the CAR further comprises a CD8 hinge region, a CD8 transmembrane region, and a CD3ζ intracellular region. According to claim 1 or 2,
An isolated nucleic acid molecule, wherein the isolated nucleic acid molecule comprises a first polynucleotide encoding CAR and a second polynucleotide encoding IL-15. According to any one of claims 1 to 3,
An isolated nucleic acid molecule, further comprising a third polynucleotide encoding CCL19. According to any one of claims 1 to 4,
An isolated nucleic acid molecule, wherein said IL-15 is human IL-15. According to any one of claims 1 to 5,
An isolated nucleic acid molecule, wherein said CCL19 is human CCL19. According to any one of claims 1 to 6,
The antibody comprises a heavy chain variable region (VH) and a light chain variable region (VL), wherein the VH comprises three complementarity-determining regions (CDRs) comprising SEQ ID NOs: 16-18, and the VL comprises SEQ ID NOs: 19-19. An isolated nucleic acid molecule containing 3 CDRs, including 21. According to any one of claims 1 to 7,
An isolated nucleic acid molecule, wherein the VH comprises SEQ ID NO: 22 and the VL comprises SEQ ID NO: 23. According to any one of claims 1 to 8,
An isolated nucleic acid molecule, wherein the antibody comprises a single-chain variable fragment (scFv) format. According to any one of claims 1 to 9,
An isolated nucleic acid molecule, wherein the antibody comprises SEQ ID NO: 1. According to any one of claims 1 to 10,
An isolated nucleic acid molecule, wherein the 4-1BB intracellular region comprises SEQ ID NO:24. According to paragraph 2,
An isolated nucleic acid molecule, wherein the CD3ζ intracellular region comprises SEQ ID NO:25. According to claim 2 or 12,
An isolated nucleic acid molecule, wherein the 4-1BB intracellular region is upstream of the CD3ζ intracellular region in the isolated nucleic acid molecule. According to paragraph 2,
An isolated nucleic acid molecule, wherein the CD8 hinge region comprises SEQ ID NO:26. According to paragraph 2,
An isolated nucleic acid molecule, wherein the CD8 transmembrane region comprises SEQ ID NO:27. According to claim 2 or 14,
An isolated nucleic acid molecule, wherein the CAR further comprises a peptide linker of 3 to 10 amino acid residues in length connecting the antibody and the CD8 hinge region. According to clause 16,
An isolated nucleic acid molecule, wherein the peptide linker comprises SEQ ID NO:4. According to any one of claims 1 to 17,
An isolated nucleic acid molecule, wherein the CAR further comprises a signaling peptide. According to clause 18,
An isolated nucleic acid molecule, wherein the signaling peptide is located upstream of an antibody that specifically recognizes human mesothelin in the isolated nucleic acid molecule. According to claim 18 or 19,
An isolated nucleic acid molecule, wherein the signaling peptide comprises SEQ ID NO:2. According to any one of claims 1 to 20,
An isolated nucleic acid molecule, wherein a second polynucleotide encoding IL-15 and a third polynucleotide optionally encoding CCL19 are each independently transcribed under a promoter comprising a polynucleotide encoding a self-cleaving 2A peptide (2A peptide). . According to clause 21,
An isolated nucleic acid molecule, wherein said 2A peptide comprises SEQ ID NO:5. According to any one of claims 1 to 22,
An isolated nucleic acid molecule, wherein said IL-15 comprises a sequence selected from SEQ ID NOs: 8-11. According to any one of claims 1 to 22,
An isolated nucleic acid molecule, wherein said IL-15 comprises a sequence selected from SEQ ID NO: 28 or 29. According to any one of claims 1 to 24,
An isolated nucleic acid molecule wherein said CCL19 comprises SEQ ID NO:13. According to any one of claims 1 to 25,
The first polynucleotide encoding CAR and the second polynucleotide encoding IL-15 are arranged in a nucleic acid molecule from 5' end to 3' end, the first polynucleotide encoding CAR - the second polynucleotide encoding IL-15. An isolated nucleic acid molecule that is arranged like a polynucleotide. According to any one of claims 1 to 25,
A first polynucleotide encoding CAR, a second polynucleotide encoding IL-15, and a third polynucleotide encoding CCL19 are arranged in a nucleic acid molecule from the 5' end to the 3' end, the first polynucleotide encoding CAR. An isolated nucleic acid molecule arranged with a nucleotide - a second polynucleotide encoding IL-15 - a third polynucleotide encoding CCL19. According to any one of claims 1 to 27,
An isolated nucleic acid molecule, wherein the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO: 14 or 31. According to any one of claims 1 to 27,
An isolated nucleic acid molecule, wherein the isolated nucleic acid molecule encodes a polypeptide comprising SEQ ID NO: 34, 35, 36, or 15. According to any one of claims 1 to 27,
An isolated nucleic acid molecule comprising SEQ ID NO: 37, 38, 39, or 40. A vector containing the nucleic acid molecule of any one of claims 1 to 30. According to clause 31,
A vector, wherein the vector is a viral vector, optionally an expression vector. According to clause 32,
A vector, wherein the viral vector is selected from retroviral vectors, lentiviral vectors, adenoviral vectors and adeno-associated virus (AAV) vectors. According to claim 31 or 32,
A vector, wherein the viral vector is a pSFG vector, pMSGV vector, or pMSCV vector. According to clause 31,
A vector, wherein the vector is a plasmid. An immune cell comprising the nucleic acid molecule of any one of claims 1 to 30 or the vector of any of claims 31 to 35. According to clause 36,
An immune cell, wherein the immune cell further comprises a polynucleotide encoding a gamma-TCR (γTCR) and a polynucleotide encoding a delta-TCR (δTCR). According to clause 36,
An immune cell wherein the γTCR is V gamma 9 TCR (Vγ9 TCR) and the δTCR is V delta 2 TCR (Vδ2TCR). Immune cells that express:
a) a chimeric antigen receptor (CAR) comprising an antibody that specifically recognizes human mesothelin and the 4-1BB intracellular region; and
b) IL-15; and
c) Optionally CCL19. According to clause 39,
An immune cell, wherein the CAR further comprises a CD8 hinge region, a CD8 transmembrane region and a CD3ζ intracellular region. According to any one of claims 36 to 40,
An immune cell, wherein the immune cell is a T cell, natural killer (NK) cell, B cell, antigen presenting cell, or granulocyte, optionally a T cell or NK cell. According to any one of claims 36 to 41,
Immune cells, wherein the immune cells are derived from induced pluripotent stem cells (iPSC). According to any one of claims 36 to 42,
An immune cell, wherein the immune cell does not express an alpha TCR (αTCR) and/or a beta TCR (βTCR), optionally an αβTCR. According to any one of claims 36 to 43,
An immune cell, wherein the immune cell expresses γδTCR. According to clause 44,
An immune cell, wherein the γδTCR includes Vγ9 TCR and Vδ2TCR. A pharmaceutical composition comprising the immune cell of any one of claims 35 to 44 and a pharmaceutically acceptable excipient. A method of treating a mesothelin-expressing cancer, comprising administering the immune cell of any one of claims 35 to 44 or the pharmaceutical composition of claim 46 to a subject in need of treatment. According to clause 47,
The mesothelin-expressing cancer may optionally be mesothelioma, colorectal cancer, pancreatic cancer, thymic cancer, bile duct cancer, lung cancer, skin cancer, breast cancer, prostate cancer, urinary bladder cancer, vaginal cancer, cervical cancer, uterine cancer, liver cancer, kidney cancer, spleen cancer, tracheal cancer, or bronchial cancer. A solid tumor selected from cancer, stomach cancer, esophageal cancer, gallbladder cancer, testicular cancer, ovarian cancer, and bone cancer. According to clause 47,
The method of claim 1, wherein the mesothelin-expressing cancer is a hematopoietic cancer. According to clause 47,
The method of claim 1, wherein the mesothelin-expressing cancer is a sarcoma optionally selected from chondrosarcoma, Ewing's sarcoma, malignant hemangioendothelioma, malignant schwannoma, osteosarcoma, and soft tissue sarcoma. According to any one of claims 47 to 50,
The method of claim 1, wherein the mesothelin-expressing cancer is a metastatic cancer. According to any one of claims 47 to 50,
The method of claim 1, wherein the mesothelin-expressing cancer is a relapsed cancer or a refractory cancer. The method according to any one of claims 47 to 52,
The method further comprises administering an additional therapeutic agent or additional therapeutic regimen to the subject. According to clause 53,
The method of claim 1, wherein the additional therapeutic agent comprises a chemotherapy agent, an immunotherapy agent, a targeted therapy, radiation therapy, or a combination thereof. According to clause 53,
The method of claim 1, wherein the additional therapeutic regimen comprises first line therapy. According to clause 53,
The method of claim 1, wherein the additional therapeutic regimen comprises surgery. The method according to any one of claims 53 to 56,
A method, wherein the immune cell of any one of claims 36 to 44 or the pharmaceutical composition of claim 45 and an additional therapeutic agent are administered simultaneously. The method according to any one of claims 52 to 55,
A method, wherein the immune cell of any one of claims 36 to 44 or the pharmaceutical composition of claim 45 and an additional therapeutic agent are administered sequentially. According to clause 57,
A method, wherein the immune cells of any one of claims 36 to 44 or the pharmaceutical composition of claim 5 are administered to the subject prior to administration of an additional therapeutic agent. According to clause 57,
A method, wherein the immune cells of any one of claims 35 to 43 or the pharmaceutical composition of claim 45 are administered to the subject following administration of an additional therapeutic agent. According to any one of claims 46 to 59,
A method, wherein the subject is a human. A method of reducing tumor cell proliferation, comprising:
A method comprising contacting a tumor cell with the immune cell of any one of claims 36 to 45 to reduce tumor cell proliferation. According to clause 62,
A method, wherein the method is an in vitro method. According to clause 62,
A method, wherein the method is an in vivo method. A method for producing immune cells expressing a cell surface molecule that specifically recognizes human mesothelin, IL-15, and optionally CCL19, comprising:
A cell surface molecule that specifically recognizes human mesothelin by immune cells by introducing the nucleic acid molecule of any one of claims 1 to 30 or the vector of any of claims 31 to 35 into immune cells, A method comprising inducing expression of IL-15 and optionally CCL19. According to clause 65,
The method of claim 1, wherein the immune cells are T cells, natural killer (NK) cells, B cells, antigen presenting cells, or granulocytes, optionally T cells or NK cells. The nucleic acid molecule of any one of claims 1 to 30; A kit, comprising the vector of any one of claims 31 to 35, the immune cell of any of claims 36 to 45, or the pharmaceutical composition of claim 46, and instructions for use.

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